Compositions and methods for controlling Leptinotarsa

ABSTRACT

Disclosed herein are methods of controlling insect pests, in particular  Leptinotarsa  spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with improved resistance to infestation by  Leptinotarsa  spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of  Leptinotarsa  spp.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCELISTINGS

This application is a Continuation of U.S. application Ser. No.14/608,951, filed on 29 Jan. 2015, which is a Continuation of U.S.application Ser. No. 14/335,135, filed on 18 Jul. 2014, which claimspriority to U.S. Provisional Patent Application No. 61/856,137 filed 19Jul. 2013, U.S. Provisional Patent Application No. 61/899,000 filed 1Nov. 2013, and U.S. Provisional Patent Application No. 61/980,800 filed17 Apr. 2014, which are incorporated by reference in their entiretyherein. The sequence listings contained in the files “40-21_60191_A.txt”(2,291 kilobytes, created on 19 Jul. 2013, filed with U.S. ProvisionalPatent Application No. 61/856,137 on 19 Jul. 2013), “40-21_60191_0001US_ST25.txt” (2,322 kilobytes, created on 30 Oct. 2013, filed with U.S.Provisional Patent Application No. 61/899,000 on 1 Nov. 2013), and“40-21_60191_0002_US_ST25.txt” (2,338 kilobytes, created on 17 Apr.2014, filed with U.S. Provisional Patent Application No. 61/980,800 on17 Apr. 2014) are incorporated by reference in their entirety herein.The sequence listing contained in the file “P34157US07_SEQ.txt”(2,532,942 bytes, created on Nov. 21, 2017) is filed herewith andincorporated by reference in its entirety herein.

FIELD

Methods for controlling invertebrate pest infestations, particularly inplants, as well as compositions, polynucleotides, and recombinant DNAconstructs useful in such methods are disclosed. More specifically, thisinvention is related to polynucleotides and methods of use thereof formodifying the expression of genes in an insect pest, particularlythrough RNA interference. Pest species of interest include Leptinotarsaspecies, especially those that infest crop plants.

BACKGROUND

Commercial crops are often the targets of attack by invertebrate pestssuch as insects. Compositions for controlling insect infestations inplants have typically been in the form of chemical insecticides.However, there are several disadvantages to using chemical insecticides.For example, chemical insecticides are generally not selective, andapplications of chemical insecticides intended to control insect pestsin crop plants can exert their effects on non-target insects and otherinvertebrates as well. Chemical insecticides often persist in theenvironment and can be slow to degrade, thus potentially accumulating inthe food chain. Furthermore the use of persistent chemical insecticidescan result in the development of resistance in the target insectspecies. Thus there has been a long felt need for more environmentallyfriendly methods for controlling or eradicating insect infestation on orin plants, i.e., methods which are species-selective, environmentallyinert, non-persistent, and biodegradable, and that fit well into pestresistance management schemes.

RNA interference (RNAi, RNA-mediated gene suppression) is anotherapproach used for pest control. In invertebrates, RNAi-based genesuppression was first demonstrated in nematodes (Fire et al., (1998)Nature, 391:806-811; Timmons & Fire (1998) Nature, 395:854).Subsequently, RNAi-based suppression of invertebrate genes usingrecombinant nucleic acid techniques has been reported in a number ofspecies, including agriculturally or economically important pests fromvarious insect and nematode taxa.

Leptinotarsa spp. form a genus including a number of species that infestcommercially important plants, including many solanaceous plants (e.g.,potato, tomato, eggplant, peppers, tobacco, and petunia). For example,Leptinotarsa decemlineata (Colorado potato beetle, CPB) is an early- tomid-season pest affecting solanaceous plants such as potato. Coloradopotato beetles primarily feed on above-ground portions of the plant, anddefoliation leads to lower tuber yields. Methods and compositions forcontrolling insect pests, in particular Leptinotarsa spp. which infestcrop plants are desired.

SUMMARY

The present embodiments are related to control of Leptinotarsa species,especially those that are economically or agriculturally importantpests. In various embodiments, the Leptinotarsa species is at least oneselected from the group consisting of Leptinotarsa behrensi,Leptinotarsa collinsi, Leptinotarsa decemlineata (Colorado potatobeetle), Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman's greenpotato beetle), Leptinotarsa heydeni, Leptinotarsa juncta (false potatobeetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsapeninsularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsatlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica. Inspecific embodiments, the Leptinotarsa species is at least one selectedfrom the group consisting of Leptinotarsa decemlineata (Colorado potatobeetle), Leptinotarsa juncta (false potato beetle), Leptinotarsahaldemani (Haldeman's green potato beetle), and Leptinotarsa lineolata(burrobrush leaf beetle).

The compositions and methods described herein include recombinantpolynucleotide molecules, such as recombinant DNA constructs for makingtransgenic plants resistant to infestation by Leptinotarsa species, andsingle- or double-stranded DNA or RNA molecules, referred to herein as“triggers”, that are useful for controlling or preventing infestation ofa plant by that Leptinotarsa species. In some embodiments,polynucleotide triggers are provided as topically applied agents forcontrolling or preventing infestation of a plant by a Leptinotarsaspecies. In some embodiments, solanaceous plants with improvedresistance to infestation by Leptinotarsa species, such as transgenicsolanaceous plants (including seeds or propagatable parts such astubers) expressing a polynucleotide trigger are provided. In someembodiments, solanaceous plants (including seeds or propagatable partssuch as tubers) that have been topically treated with a compositioncomprising a polynucleotide trigger (e.g., solanaceous plants that havebeen sprayed with a solution of dsRNA molecules) are provided. Alsoprovided are polynucleotide-containing compositions that are topicallyapplied to a Leptinotarsa species or to a plant, plant part, or seed tobe protected from infestation by a Leptinotarsa species.

Several embodiments relate to suppression of a target gene in aLeptinotarsa species by a polynucleotide trigger. Some embodimentsrelate to methods for selecting Leptinotarsa target genes that arelikely to be effective targets for RNAi-mediated control of aLeptinotarsa species. In some embodiments, target genes selected forRNAi-mediated suppression are genes that are non-repetitive andnon-redundant in a Leptinotarsa species genome, or that have lownucleotide diversity, or that are evolutionarily or functionallyconstrained to have a more synonymous (K_(s)) than nonsynonymous (K_(a))nucleotide changes. Provided herein are nucleotide sequences referred toherein as the “Target Gene Sequences Group”, which consists of SEQ IDNOs:1-725 and SEQ ID NOs:726-830 and SEQ ID NOs:1087-1094. Also providedare nucleotide sequences referred to herein as the “Trigger SequencesGroup”, which consists of SEQ ID NOs:831, 842, 849, 898, 910, 925, 928,931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950,951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970,971, 973, 976, 978, 979, 982, 983, 985, 987, 988, 989, 991, 992, 994,995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019,1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040,1041, 1042, 1043, 1045, 1046, 1047, 1049, 1050, 1053, 1054, 1058, 1060,1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078,1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101,1102, 1103, 1104, 1105, 1110, 1111, 1112, 1113, and 1114.

In one aspect, a method for controlling a Leptinotarsa speciesinfestation of a plant comprising contacting the Leptinotarsa specieswith a polynucleotide comprising at least one segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity (e.g., a segment of 21 contiguous nucleotides with a sequenceof 100% identity) with a corresponding fragment of a DNA having asequence selected from the group consisting of: the Target GeneSequences Group, or the DNA complement thereof. In an embodiment, themethod for controlling a Leptinotarsa species infestation of a plantcomprises contacting the Leptinotarsa species with a polynucleotidecomprising a nucleotide sequence that is complementary to at least 21contiguous nucleotides of a target gene having a nucleotide sequenceselected from the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094, or an RNA transcribed from thetarget gene. In some embodiments, the polynucleotide is double-strandedRNA. In some embodiments, the polynucleotide comprises one or morenucleotide sequences selected from the Trigger Sequences Group. In someembodiments, the contacting with a polynucleotide is achieved by topicalapplication of the polynucleotide, or of a composition or solutioncontaining the polynucleotide (e.g., by spraying or dusting or soaking),directly to the Leptinotarsa species or to a surface or matrix (e.g., aplant or soil) contacted by the Leptinotarsa species. In someembodiments, the contacting with a polynucleotide is achieved byproviding a polynucleotide that is ingested by the Leptinotarsa species.In some embodiments, the contacting with a polynucleotide is achieved byproviding a transgenic plant that expresses to the Leptinotarsa species.

Several embodiments relate to a method for controlling a Leptinotarsaspecies infestation of a plant by providing in the diet of aLeptinotarsa species an agent comprising a polynucleotide having atleast one segment of 18 or more contiguous nucleotides with a sequenceof about 95% to about 100% identity (e.g., a segment of 21 contiguousnucleotides with a sequence of 100% identity) with a correspondingfragment of a DNA having a sequence selected from the group consistingof: the Target Gene Sequences Group, or the DNA complement thereof, andwherein the agent functions upon ingestion by the Leptinotarsa speciesto inhibit a biological function within the Leptinotarsa species therebycontrolling infestation by the Leptinotarsa species. In an embodiment,the method for controlling a Leptinotarsa species infestation of a plantcomprises providing in the diet of the Leptinotarsa species apolynucleotide comprising a nucleotide sequence that is complementary toat least 21 contiguous nucleotides of a target gene having a nucleotidesequence selected from the group consisting of: SEQ ID NO:730, SEQ IDNO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094, or an RNA transcribed from thetarget gene. In some embodiments, the polynucleotide comprises one ormore nucleotide sequences selected from the Trigger Sequences Group. Insome embodiments, the polynucleotide is double-stranded RNA. In someembodiments, the agent containing the polynucleotide is formulated forapplication to fields of crop plants, e.g., in sprayable solutions oremulsions, tank mixes, or powders. In some embodiments, the agent isbiologically produced, e.g., in the form of a microbial fermentationproduct or expressed in a transgenic plant cell.

In another aspect, a method of causing mortality or stunting inLeptinotarsa species larvae is provided. In some embodiments, at leastone RNA comprising at least one silencing element is provided in thediet of a Leptinotarsa species larvae wherein ingestion of the RNA bythe Leptinotarsa species larvae results in mortality or stunting in theLeptinotarsa species larvae. In some embodiments, the silencing elementis essentially identical or essentially complementary to a fragment of atarget gene sequence of the Leptinotarsa species larvae, wherein thetarget gene is selected from the group consisting of the genes in theTarget Gene Sequences Group In an embodiment, the method of causingmortality or stunting in larvae of the Leptinotarsa species comprisesproviding in the diet of the larvae at least one polynucleotidecomprising at least one silencing element comprising 21 contiguousnucleotides that are complementary to a target gene having a nucleotidesequence selected from the group consisting of: SEQ ID NO:730, SEQ IDNO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094, or an RNA transcribed from thetarget gene. In some embodiments, the silencing element comprises one ormore nucleotide sequences selected from the Trigger Sequences Group. Insome embodiments, the polynucleotide is double-stranded RNA. Someembodiments relate to a method of causing mortality or lower fecundityin Leptinotarsa species comprising providing in the diet of Leptinotarsaspecies at least one RNA comprising at least one silencing elementessentially identical or essentially complementary to a fragment of atarget gene sequence of the Leptinotarsa species larvae whereiningestion of the RNA by the Leptinotarsa species results in mortality orlower fecundity in the Leptinotarsa species. In some embodiments, thetarget gene is selected from the group consisting of the genes in theTarget Gene Sequences Group. In some embodiments, the method causes adecrease in metamorphosis rate or a decrease in feeding activity. Insome embodiments, the method is useful for providing plants havingincreased resistance to infestation by Leptinotarsa species.

Several embodiments relate to a method of providing a plant havingimproved resistance to a Leptinotarsa species infestation comprisingtopically applying to the plant a composition comprising at least onepolynucleotide having at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity (e.g., asegment of 21 contiguous nucleotides with a sequence of 100% identity)with a corresponding fragment of a DNA having a sequence selected fromthe group consisting of: the Target Gene Sequences Group, or the DNAcomplement thereof. In an embodiment, the method of providing a planthaving improved resistance to a Leptinotarsa species infestationcomprises topically applying to the plant a composition comprising atleast one polynucleotide comprising a nucleotide sequence that iscomplementary to at least 21 contiguous nucleotides of a target genehaving a nucleotide sequence selected from the group consisting of: SEQID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQ IDNOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs:1087-1094, or an RNAtranscribed from the target gene. In an embodiment, the method ofproviding a plant having improved resistance to a Leptinotarsa speciesinfestation comprises topically applying to the plant a compositioncomprising at least one polynucleotide in a manner such that aneffective amount of the polynucleotide is ingested by a Leptinotarsaspecies feeding on the plant, the polynucleotide comprising at least 21contiguous nucleotides that are complementary to a target gene having anucleotide sequence selected from the group consisting of: SEQ IDNO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQ IDNOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs:1087-1094, or an RNAtranscribed from the target gene. In some embodiments, thepolynucleotide comprises one or more nucleotide sequences selected fromthe Trigger Sequences Group. In some embodiments, the polynucleotide isdouble-stranded RNA. Several embodiments relate to compositionscomprising the polynucleotide, formulated for application to fields ofcrop plants, e.g., in sprayable solutions or emulsions, tank mixes, orpowders.

Several embodiments relate to an insecticidal composition forcontrolling a Leptinotarsa species comprising an insecticidallyeffective amount of at least one polynucleotide molecule comprising atleast one segment of 18 or more contiguous nucleotides that areessentially identical or complementary (e.g., a segment of 21 contiguousnucleotides with a sequence of 100% identity or complementarity) withthe corresponding fragment of a DNA having a sequence selected from thegroup consisting of: the Target Gene Sequences Group, or the DNAcomplement thereof. In some embodiments, the polynucleotide moleculecomprises at least 21 contiguous nucleotides that are complementary to atarget gene having a nucleotide sequence selected from the groupconsisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ IDNOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or an RNA transcribed from the target gene. In someembodiments, the polynucleotide comprises one or more nucleotidesequences selected from the Trigger Sequences Group. In someembodiments, the polynucleotide molecule is a recombinantpolynucleotide. In some embodiments, the polynucleotide molecule is RNA.In some embodiments, the polynucleotide molecule is double-stranded RNA.Related embodiments include insecticidal compositions comprising thepolynucleotide molecule formulated for application to fields of cropplants, e.g., in sprayable solutions or emulsions, tank mixes, orpowders, and optionally comprising one or more additional components,such as a carrier agent, a surfactant, a cationic lipid, anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator.

Several embodiments relate to a method of providing a plant havingimproved resistance to a Leptinotarsa species infestation comprisingexpressing in the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to (e.g., a segment of 21 contiguousnucleotides with a sequence of 100% identity or complementarity with)the corresponding fragment of a DNA having a sequence selected from thegroup consisting of: the Target Gene Sequences Group, or the DNAcomplement thereof. In some embodiments, the polynucleotide comprisesone or more nucleotide sequences selected from the Trigger SequencesGroup. In some embodiments, the polynucleotide is double-stranded RNA.

Several embodiments relate to a recombinant DNA construct comprising aheterologous promoter operably linked to a DNA element comprising atleast one segment of 18 or more contiguous nucleotides with a sequenceof about 95% to about 100% identity (e.g., a segment of 21 contiguousnucleotides with a sequence of 100% identity) with the correspondingfragment of a DNA having a sequence selected from the group consistingof: the Target Gene Sequences Group, or the DNA complement thereof. Insome embodiments, the DNA element encodes a double-stranded RNA. In someembodiments, the double-stranded RNA comprises one or more nucleotidesequences selected from the Trigger Sequences Group. Related embodimentsinclude a plant chromosome or a plastid or a recombinant plant virusvector or a recombinant baculovirus vector comprising the recombinantDNA construct, or comprising the DNA element without the heterologouspromoter.

Several embodiments relate to a transgenic solanaceous plant cell havingin its genome a recombinant DNA encoding RNA that suppresses expressionof a target gene in a Leptinotarsa species that contacts or ingests theRNA, wherein the RNA comprises at least one silencing element having atleast one segment of 18 or more contiguous nucleotides complementary toa fragment of a target gene. In some embodiments, the target gene isselected from the Target Gene Sequences Group. A specific embodiment isa transgenic solanaceous plant cell having in its genome a recombinantDNA encoding RNA for silencing one or more target genes selected fromthe group consisting of exocyst genes, ribosomal protein genes, andproteosome genes. In some embodiments, the RNA comprises one or morenucleotide sequences selected from the Trigger Sequences Group.

Several embodiments relate to an isolated recombinant RNA molecule thatcauses mortality or stunting of growth in a Leptinotarsa species wheningested or contacted by the Leptinotarsa species, wherein therecombinant RNA molecule comprises at least one segment of 18 or morecontiguous nucleotides that are essentially complementary to (e.g., asegment of 21 contiguous nucleotides with a sequence of 100%complementarity with) the corresponding of a DNA having a sequenceselected from the group consisting of: the Target Gene Sequences Group,or the DNA complement thereof. In some embodiments, the recombinant RNAmolecule is double-stranded RNA. Specific embodiments include anisolated recombinant RNA molecule for suppressing expression of aribosomal protein such as a ribosomal L7 protein or a protein encoded bySEQ ID NO:730, and an isolated recombinant double-stranded RNA moleculehaving a sequence selected from the group consisting of SEQ ID NO:989,988, 1104, or 1105.

Several embodiments relate to a method of providing a plant havingimproved resistance to a Leptinotarsa species infestation comprisingproviding to the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to (e.g., a segment of 21 contiguousnucleotides with a sequence of 100% identity or complementarity with)the corresponding fragment of a target gene selected from the TargetGene Sequences Group. In an embodiment, the method of providing a planthaving improved resistance to a Leptinotarsa species infestationcomprises providing to the plant at least one polynucleotide comprisingat least one segment that is identical or complementary to at least 21contiguous nucleotides of a target gene or an RNA transcribed from thetarget gene, wherein the target gene is selected from the groupconsisting of: the genes identified in the Target Gene Sequences Group.In some embodiments, the polynucleotide comprises one or more nucleotidesequences selected from the Trigger Sequences Group. In someembodiments, the polynucleotide is double-stranded RNA.

Several embodiments relate to a method for controlling a Leptinotarsaspecies infestation of a plant comprising contacting the Leptinotarsaspecies with a polynucleotide comprising at least one segment of 18 ormore contiguous nucleotides that are essentially identical orcomplementary to (e.g., a segment of 21 contiguous nucleotides with asequence of 100% identity or complementarity with) the correspondingfragment of equivalent length of a DNA of a target gene selected fromthe Target Gene Sequences Group. In some embodiments, the polynucleotideis double-stranded RNA. In an embodiment, the method for controlling aLeptinotarsa species infestation of a plant comprises contacting theLeptinotarsa species with an effective amount of a double-stranded RNA,one strand of which is complementary to at least 21 contiguousnucleotides of a gene that encodes a ribosomal protein, wherein RNAinterference is induced and mortality occurs. In some embodiments, thedouble-stranded RNA comprises one or more nucleotide sequences selectedfrom the Trigger Sequences Group.

Several embodiments relate to a method of selecting target genes forRNAi-mediated silencing from a plant genome or from an animal genome. Invarious embodiments, the method provides a subset of target genes thatare present in single- or low-copy-number (non-repetitive andnon-redundant) in a particular genome, or that have low nucleotidediversity, or that have a ratio of synonymous (K_(s)) to nonsynonymous(K_(a)) nucleotide changes where K_(s)>>K_(a).

Several embodiments relate to man-made compositions comprising at leastone polynucleotide as described herein. In some embodiments,formulations useful for topical application to a plant or substance inneed of protection from a Leptinotarsa species infestation are provided.In some embodiments, recombinant constructs and vectors useful formaking transgenic solanaceous plant cells and transgenic solanaceousplants are provided. In some embodiments, formulations and coatingsuseful for treating solanaceous plants, solanaceous plant seeds orpropagatable parts such as tubers are provided. In some embodiments,commodity products and foodstuffs produced from such solanaceous plants,seeds, or propagatable parts treated with or containing a polynucleotideas described herein (especially commodity products and foodstuffs havinga detectable amount of a polynucleotide as described herein) areprovided. Several embodiments relate to polyclonal or monoclonalantibodies that bind a protein encoded by a sequence or a fragment of asequence selected from the Target Gene Sequences Group. Another aspectrelates to polyclonal or monoclonal antibodies that bind a proteinencoded by a sequence or a fragment of a sequence selected from theTrigger Sequences Group, or the complement thereof. Such antibodies aremade by routine methods as known to one of ordinary skill in the art.

In the various embodiments described herein, the plant can be any plantthat is subject to infestation by a Leptinotarsa species. Of particularinterest are embodiments wherein the plant is a solanaceous plant(family Solanaceae). Examples include a plant selected from the groupconsisting of potato, tomato, and eggplant. Embodiments include thosewherein the plant is an ungerminated solanaceous plant seed, asolanaceous plant in a vegetative stage, or a solanaceous plant in areproductive stage. Embodiments include those wherein the plant is a“seed potato”, meaning a potato tuber or piece of potato tuber which canbe propagated into new potato plants.

Other aspects and specific embodiments of this invention are disclosedin the following detailed description.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Where a term is provided in thesingular, the inventors also contemplate aspects of the inventiondescribed by the plural of that term. Where there are discrepancies interms and definitions used in references that are incorporated byreference, the terms used in this application shall have the definitionsgiven herein. Other technical terms used have their ordinary meaning inthe art in which they are used, as exemplified by various art-specificdictionaries, for example, “The American Heritage® Science Dictionary”(Editors of the American Heritage Dictionaries, 2011, Houghton MifflinHarcourt, Boston and New York), the “McGraw-Hill Dictionary ofScientific and Technical Terms” (6^(th) edition, 2002, McGraw-Hill, NewYork), or the “Oxford Dictionary of Biology” (6^(th) edition, 2008,Oxford University Press, Oxford and New York). The inventors do notintend to be limited to a mechanism or mode of action. Reference theretois provided for illustrative purposes only.

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. One of skill in the art would be aware that a given DNAsequence is understood to define a corresponding RNA sequence which isidentical to the DNA sequence except for replacement of the thymine (T)nucleotides of the DNA with uracil (U) nucleotides. Thus, providing aspecific DNA sequence is understood to define the exact RNA equivalent.A given first polynucleotide sequence, whether DNA or RNA, furtherdefines the sequence of its exact complement (which can be DNA or RNA),a second polynucleotide that hybridizes perfectly to the firstpolynucleotide by forming Watson-Crick base-pairs. For DNA:DNA duplexes(hybridized strands), base-pairs are adenine:thymine orguanine:cytosine; for DNA:RNA duplexes, base-pairs are adenine:uracil orguanine:cytosine. Thus, the nucleotide sequence of a blunt-endeddouble-stranded polynucleotide that is perfectly hybridized (where there“100% complementarity” between the strands or where the strands are“complementary”) is unambiguously defined by providing the nucleotidesequence of one strand, whether given as DNA or RNA. By “essentiallyidentical” or “essentially complementary” to a target gene or a fragmentof a target gene is meant that a polynucleotide strand (or at least onestrand of a double-stranded polynucleotide) is designed to hybridize(generally under physiological conditions such as those found in aliving plant or animal cell) to a target gene or to a fragment of atarget gene or to the transcript of the target gene or the fragment of atarget gene; one of skill in the art would understand that suchhybridization does not necessarily require 100% sequence identity orcomplementarity. A first nucleic acid sequence is “operably” connectedor “linked” with a second nucleic acid sequence when the first nucleicacid sequence is placed in a functional relationship with the secondnucleic acid sequence. For example, a promoter sequence is “operablylinked” to a DNA if the promoter provides for transcription orexpression of the DNA. Generally, operably linked DNA sequences arecontiguous.

The term “polynucleotide” commonly refers to a DNA or RNA moleculecontaining multiple nucleotides and generally refers both to“oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides inlength) and longer polynucleotides of 26 or more nucleotides.Polynucleotides also include molecules containing multiple nucleotidesincluding non-canonical nucleotides or chemically modified nucleotidesas commonly practiced in the art; see, e.g., chemical modificationsdisclosed in the technical manual “RNA Interference (RNAi) and DsiRNAs”,2011 (Integrated DNA Technologies Coralville, Iowa). Generally,polynucleotides as described herein, whether DNA or RNA or both, andwhether single- or double-stranded, include at least one segment of 18or more contiguous nucleotides (or, in the case of double-strandedpolynucleotides, at least 18 contiguous base-pairs) that are essentiallyidentical or complementary to a fragment of equivalent size of the DNAof a target gene or the target gene's RNA transcript. Throughout thisdisclosure, “at least 18 contiguous” means “from about 18 to about10,000, including every whole number point in between”. Thus,embodiments of this invention include oligonucleotides having a lengthof 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers,23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having alength of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 110,about 120, about 130, about 140, about 150, about 160, about 170, about180, about 190, about 200, about 210, about 220, about 230, about 240,about 250, about 260, about 270, about 280, about 290, or about 300nucleotides), or long polynucleotides having a length greater than about300 nucleotides (e.g., polynucleotides of between about 300 to about 400nucleotides, between about 400 to about 500 nucleotides, between about500 to about 600 nucleotides, between about 600 to about 700nucleotides, between about 700 to about 800 nucleotides, between about800 to about 900 nucleotides, between about 900 to about 1000nucleotides, between about 300 to about 500 nucleotides, between about300 to about 600 nucleotides, between about 300 to about 700nucleotides, between about 300 to about 800 nucleotides, between about300 to about 900 nucleotides, or about 1000 nucleotides in length, oreven greater than about 1000 nucleotides in length, for example up tothe entire length of a target gene including coding or non-coding orboth coding and non-coding portions of the target gene). Where apolynucleotide is double-stranded, its length can be similarly describedin terms of base pairs.

The polynucleotides described herein can be single-stranded (ss) ordouble-stranded (ds). “Double-stranded” refers to the base-pairing thatoccurs between sufficiently complementary, anti-parallel nucleic acidstrands to form a double-stranded nucleic acid structure, generallyunder physiologically relevant conditions. Embodiments include thosewherein the polynucleotide is selected from the group consisting ofsense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA),double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), adouble-stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; amixture of polynucleotides of any of these types can be used. In someembodiments, the polynucleotide is double-stranded RNA of a lengthgreater than that which is typical of naturally occurring regulatorysmall RNAs (such as endogenously produced siRNAs and mature miRNAs). Insome embodiments, the polynucleotide is double-stranded RNA of at leastabout 30 contiguous base-pairs in length. In some embodiments, thepolynucleotide is double-stranded RNA with a length of between about 50to about 500 base-pairs. In some embodiments, the polynucleotide caninclude components other than standard ribonucleotides, e.g., anembodiment is an RNA that comprises terminal deoxyribonucleotides.

In various embodiments, the polynucleotide described herein comprisenaturally occurring nucleotides, such as those which occur in DNA andRNA. In certain embodiments, the polynucleotide is a combination ofribonucleotides and deoxyribonucleotides, for example, syntheticpolynucleotides consisting mainly of ribonucleotides but with one ormore terminal deoxyribonucleotides or one or more terminaldideoxyribonucleotides or synthetic polynucleotides consisting mainly ofdeoxyribonucleotides but with one or more terminaldideoxyribonucleotides. In certain embodiments, the polynucleotidecomprises non-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In certain embodiments, the polynucleotide compriseschemically modified nucleotides. Examples of chemically modifiedoligonucleotides or polynucleotides are well known in the art; see, forexample, U.S. Patent Publication 2011/0171287, U.S. Patent Publication2011/0171176, U.S. Patent Publication 2011/0152353, U.S. PatentPublication 2011/0152346, and U.S. Patent Publication 2011/0160082,which are herein incorporated by reference. Illustrative examplesinclude, but are not limited to, the naturally occurring phosphodiesterbackbone of an oligonucleotide or polynucleotide which can be partiallyor completely modified with phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications, modifiednucleoside bases or modified sugars can be used in oligonucleotide orpolynucleotide synthesis, and oligonucleotides or polynucleotides can belabeled with a fluorescent moiety (e.g., fluorescein or rhodamine) orother label (e.g., biotin).

Several embodiments relate to a polynucleotide comprising at least onesegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with a fragment of equivalent length of a DNAor target gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof. In some embodiments, the contiguousnucleotides number at least 18, e.g., between 18-24, or between 18-28,or between 20-30, or between 20-50, or between 20-100, or between50-100, or between 50-500, or between 100-250, or between 100-500, orbetween 200-1000, or between 500-2000, or even greater. In someembodiments, the contiguous nucleotides number more than 18, e.g., 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g.,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 110, about 120, about 130, about 140, about 150, about 160, about170, about 180, about 190, about 200, about 210, about 220, about 230,about 240, about 250, about 260, about 270, about 280, about 290, about300, about 350, about 400, about 450, about 500, or greater than 500contiguous nucleotides. In some embodiments, the polynucleotidecomprises at least one segment of at least 21 contiguous nucleotideswith a sequence of 100% identity with a fragment of equivalent length ofa DNA or target gene having a sequence selected from the Target GeneSequences Group or the DNA complement thereof. In some embodiments, thepolynucleotide is a double-stranded nucleic acid (e.g., dsRNA) with onestrand comprising at least one segment of at least 21 contiguousnucleotides with 100% identity with a fragment of equivalent length of aDNA or target gene having a sequence selected from the Target GeneSequences Group or the DNA complement thereof; expressed as base-pairs,such a double-stranded nucleic acid comprises at least one segment of atleast 21 contiguous, perfectly matched base-pairs which correspond to afragment of equivalent length of a DNA or target gene having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof. In some embodiments, each segment contained in thepolynucleotide is of a length greater than that which is typical ofnaturally occurring regulatory small RNAs, for example, each segment isat least about 30 contiguous nucleotides (or base-pairs) in length. Insome embodiments, the total length of the polynucleotide, or the lengthof each segment contained in the polynucleotide, is less than the totallength of the DNA or target gene having a sequence selected from theTarget Gene Sequences Group. In some embodiments, the total length ofthe polynucleotide is between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Embodiments include those in which the polynucleotide expressed in theplant is an RNA comprising a segment having a sequence selected from thegroup consisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114, orthe complement thereof, or is an RNA hairpin encoded by a sequenceselected from the group consisting of SEQ ID NOs:1105-1109. In someembodiments, the polynucleotide is expressed in a plant. In someembodiments, the polynucleotide is topically provided to the surface ofa plant or Leptinotarsa species.

Several embodiments relate to polynucleotides that are designed tomodulate expression by inducing regulation or suppression of aLeptinotarsa species target gene. In some embodiments, thepolynucleotides are designed to have a nucleotide sequence essentiallyidentical or essentially complementary to the nucleotide sequence of aLeptinotarsa species target gene or cDNA (e.g., The Target GeneSequences Group) or to the sequence of RNA transcribed from aLeptinotarsa species target gene, which can be coding sequence ornon-coding sequence. These effective polynucleotide molecules thatmodulate expression may be referred to herein as a “polynucleotide”,“polynucleotide trigger”, “trigger”, or “triggers”.

Effective polynucleotides of any size can be used, alone or incombination, in the various methods and compositions described herein.In some embodiments, a single polynucleotide trigger is used to make acomposition (e.g., a composition for topical application, or arecombinant DNA construct useful for making a transgenic plant). Inother embodiments, a mixture or pool of different polynucleotidetriggers is used; in such cases the polynucleotide triggers can be for asingle target gene or for multiple target genes.

As used herein, the term “isolated” refers to separating a molecule fromother molecules normally associated with it in its native or naturalstate. The term “isolated” thus may refer to a DNA molecule that hasbeen separated from other DNA molecule(s) which normally are associatedwith it in its native or natural state. Such a DNA molecule may bepresent in a recombined state, such as a recombinant DNA molecule. Thus,DNA molecules fused to regulatory or coding sequences with which theyare not normally associated, for example as the result of recombinanttechniques, are considered isolated, even when integrated as a transgeneinto the chromosome of a cell or present with other DNA molecules.

As used herein, the term “Target Gene Sequences Group” refers to thegroup of sequences consisting of SEQ ID NOs:1-725 and SEQ ID NOs:726-830and SEQ ID NOs:1087-1094). As used herein, the term “Trigger SequencesGroup” refers to the group of sequences consisting of SEQ ID NOs:831,842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943,944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961,964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985,987, 988, 989, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008,1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035,1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1049,1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070,1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096,1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1110, 1111, 1112,1113, and 1114.

In various embodiments, the Leptinotarsa species is at least oneselected from the group consisting of Leptinotarsa behrensi,Leptinotarsa collinsi, Leptinotarsa decemlineata (Colorado potatobeetle), Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman's greenpotato beetle), Leptinotarsa heydeni, Leptinotarsa juncta (false potatobeetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsapeninsularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsatlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica. Inspecific embodiments, the Leptinotarsa species is at least one selectedfrom the group consisting of Leptinotarsa decemlineata (Colorado potatobeetle), Leptinotarsa juncta (false potato beetle), Leptinotarsahaldemani (Haldeman's green potato beetle), and Leptinotarsa lineolata(burrobrush leaf beetle).

Controlling Leptinotarsa Infestations by Contacting with aPolynucleotide

Provided herein are methods for controlling a Leptinotarsa speciesinfestation of a plant by contacting the Leptinotarsa species with apolynucleotide comprising at least one segment of 18 or more contiguousnucleotides having about 95% to about 100% identity or complementaritywith a corresponding fragment of a DNA or target gene selected from thegroup consisting of: the Target Gene Sequences Group, or the DNAcomplement thereof. In an embodiment, the method for controlling aLeptinotarsa species infestation of a plant comprises contacting theLeptinotarsa species with a polynucleotide comprising at least 21contiguous nucleotides with 100% identity with a corresponding fragmentof a target gene having a DNA sequence selected from the groupconsisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ IDNOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or the DNA complement thereof. In some embodiments, thepolynucleotide is a double-stranded RNA. In some embodiments, thepolynucleotide (e.g., double-stranded RNA) is chemically synthesized oris produced by expression in a microorganism or by expression in a plantcell. Embodiments include those in which the polynucleotide is a dsRNAcomprising a sequence selected from the group consisting of SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the polynucleotide is encoded by a sequence selected from thegroup consisting of SEQ ID NOs:1105-1109. In an embodiment, the methodfor controlling a Leptinotarsa species infestation of a plant comprisescontacting the Leptinotarsa species with a polynucleotide comprising anucleotide sequence that is complementary to at least 21 contiguousnucleotides of a target gene encoded by a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or an RNA transcribed from the target gene.Embodiments include those in which the polynucleotide is a dsRNAcomprising a strand having a sequence selected from the TriggerSequences Group. In some embodiments, the method uses a polynucleotidecomprising one segment of 127 contiguous nucleotides (SEQ ID NO:831)which is the anti-sense (reverse complement) sequence of 127 contiguousnucleotides of the target gene encoded by SEQ ID NO:825. In someembodiments, the method uses a polynucleotide comprising segments of 409and 403 contiguous nucleotides (SEQ ID NO:937 and SEQ ID NO:938,respectively) which are the anti-sense (reverse complement) sequences of409 and 403 contiguous nucleotides, respectively, of a target geneencoded by SEQ ID NO:732. Polynucleotides of use in the method can bedesigned for multiple target genes. Related aspects of the inventioninclude isolated polynucleotides of use in the method and plants havingimproved Leptinotarsa resistance provided by the method.

In some embodiments, the contiguous nucleotides have a sequence of about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identitywith a fragment of equivalent length of a DNA or target gene having asequence selected from the group consisting of: SEQ ID NOs:1-725 and SEQID NOs:726-830 and SEQ ID NOs:1087-1094 or the DNA complement thereof.In some embodiments, the contiguous nucleotides are exactly (100%)identical to a fragment of equivalent length of a DNA or target genehaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof. In some embodiments, the polynucleotide has anoverall sequence of about 95%, about 96%, about 97%, about 98%, about99%, or about 100% identity with a fragment of equivalent length of aDNA or target gene having a sequence selected from the Target GeneSequences Group or the DNA complement thereof. In an embodiment, thepolynucleotide comprises at least one segment of 21 contiguousnucleotides with 100% identity with the corresponding fragment of atarget gene having a DNA sequence selected from the group consisting of:SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQID NOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs:1087-1094, or the DNAcomplement thereof. In some embodiments, the polynucleotide comprises“neutral” sequence (sequence having no sequence identity orcomplementarity to the target gene) in addition to one or more segmentsof 21 contiguous nucleotides with 100% identity with the correspondingfragment of the target gene, and therefore the polynucleotide as a wholeis of much lower overall sequence identity with a target gene.

Several embodiments relate to a polynucleotide designed to suppress oneor more genes (“target genes”). The term “gene” refers to any portion ofa nucleic acid that provides for expression of a transcript or encodes atranscript. A “gene” can include, but is not limited to, a promoterregion, 5′ untranslated regions, transcript encoding regions that caninclude intronic regions, 3′ untranslated regions, or combinations ofthese regions. In some embodiments, the target genes can include codingor non-coding sequence or both. In other embodiments, the target genehas a sequence identical to or complementary to a messenger RNA, e.g.,in some embodiments the target gene is a cDNA. In specific embodiments,the polynucleotide is designed to suppress one or more target genes,where each target gene is encoded by a DNA sequence selected from theTarget Gene Sequences Group. In various embodiments, the polynucleotideis designed to suppress one or more target genes, where each target geneis encoded by a sequence selected from the Target Gene Sequences Group,and can be designed to suppress multiple target genes from this group,or to target different regions of one or more of these target genes. Inan embodiment, the polynucleotide comprises multiple segments of 21contiguous nucleotides with 100% identity with a fragment of equivalentlength of a DNA or target gene having a sequence selected from theTarget Gene Sequences Group or the DNA complement thereof. In suchcases, each segment can be identical or different in size or insequence, and can be sense or anti-sense relative to the target gene.For example, in one embodiment the polynucleotide comprises multiplesegments in tandem or repetitive arrangements, wherein each segmentcomprises 21 contiguous nucleotides with a sequence of 100% identitywith a fragment of equivalent length of a DNA or target gene having asequence selected from the Target Gene Sequences Group or the DNAcomplement thereof. In some embodiments, the segments can be fromdifferent regions of the target gene, e.g., the segments can correspondto different exon regions of the target gene. In some embodiments,“spacer” nucleotides which do not correspond to a target gene canoptionally be used in between or adjacent to the segments.

The total length of the polynucleotide of use in this method can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with a fragment of equivalent length of a DNA ortarget gene having a sequence selected from the group consisting of: theTarget Gene Sequences Group or the DNA complement thereof. In otherwords, the total length of the polynucleotide can be greater than thelength of the section or segment of the polynucleotide designed tosuppress one or more target genes, where each target gene has a DNAsequence selected from the group consisting of the Target Gene SequencesGroup. For example, the polynucleotide can have nucleotides flanking the“active” segment of at least one segment of 18 or more contiguousnucleotides that suppresses the target gene, or include “spacer”nucleotides between active segments, or can have additional nucleotidesat the 5′ end, or at the 3′ end, or at both the 5′ and 3′ ends. In anembodiment, the polynucleotide can include additional nucleotides thatare not specifically related (having a sequence not complementary oridentical to) to the DNA or target gene having a sequence selected fromthe group consisting of: the Target Gene Sequences Group or the DNAcomplement thereof, e.g., nucleotides that provide stabilizing secondarystructure or for convenience in cloning or manufacturing. In anembodiment, the polynucleotide can include additional nucleotideslocated immediately adjacent to one or more segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity with a fragment of equivalent length of a DNA or target genehaving a sequence selected from the group consisting of: the Target GeneSequences Group or the DNA complement thereof. In an embodiment, thepolynucleotide comprises one such segment, with an additional 5′ G or anadditional 3′ C or both, adjacent to the segment. In another embodiment,the polynucleotide is a double-stranded RNA comprising additionalnucleotides to form an overhang, for example, a dsRNA comprising 2deoxyribonucleotides to form a 3′ overhang. Thus in various embodiments,the nucleotide sequence of the entire polynucleotide is not 100%identical or complementary to a sequence of contiguous nucleotides inthe DNA or target gene having a sequence selected from the groupconsisting of: the Target Gene Sequences Group, or the DNA complementthereof. For example, in some embodiments the polynucleotide comprisesat least two segments each of 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of a DNA having a sequence selectedfrom the group consisting of: the Target Gene Sequences Group, or theDNA complement thereof, wherein (1) the at least two segments areseparated by one or more spacer nucleotides, or (2) the at least twosegments are arranged in an order different from that in which thecorresponding fragments occur in the DNA having a sequence selected fromthe group consisting of: the Target Gene Sequences Group, or the DNAcomplement thereof.

The polynucleotide of use in this method is provided by suitable meansknown to one in the art. Embodiments include those wherein thepolynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation.

In some embodiments the polynucleotide of use in this method is providedas an isolated DNA or RNA fragment. In some embodiments thepolynucleotide of use in this method is not part of an expressionconstruct and is lacking additional elements such as a promoter orterminator sequences). Such polynucleotides can be relatively short,such as single- or double-stranded polynucleotides of between about 18to about 300 or between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or between about 18 to about 300 orbetween about 50 to about 500 base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Embodiments include those in which the polynucleotide is a dsRNAcomprising a segment having a sequence selected from the groupconsisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114, or thecomplement thereof, or wherein the polynucleotide is encoded by asequence selected from the group consisting of SEQ ID NOs:1105-1109.Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. In some embodimentssuch recombinant expression constructs or vectors are designed toinclude additional elements, such as expression cassettes for expressinga gene of interest (e.g., an insecticidal protein).

In various embodiments of the method, the contacting comprisesapplication to a surface of the Leptinotarsa species of a suitablecomposition comprising the polynucleotide of use in this method; such acomposition can be provided, e.g., as a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or as a seed treatment. The contacting can be in theform of a seed treatment, or in the form of treatment of “seed potato”tubers or pieces of tuber (e.g., by soaking, coating, or dusting theseed potato). Suitable binders, inert carriers, surfactants, and thelike can optionally be included in the composition, as is known to oneskilled in formulation of pesticides and seed treatments. In someembodiments, the contacting comprises providing the polynucleotide in acomposition that further comprises one or more components selected fromthe group consisting of a carrier agent, a surfactant, a cationic lipid(such as that disclosed in Example 18 of U.S. patent applicationpublication 2011/0296556, incorporated by reference herein), anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator.In some embodiments, the contacting comprises providing thepolynucleotide in a composition that further comprises at least onepesticidal agent selected from the group consisting of a patatin, aplant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidalprotein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidalprotein, a Bacillus laterosporous insecticidal protein, and a Bacillussphaericus insecticidal protein. In one embodiment the contactingcomprises providing the polynucleotide in a composition that can beingested or otherwise absorbed internally by the Leptinotarsa species.

It is anticipated that the combination of certain polynucleotides of usein this method (e.g., the polynucleotide triggers described in theworking Examples) with one or more non-polynucleotide pesticidal agentswill result in a synergetic improvement in prevention or control ofLeptinotarsa species infestations, when compared to the effect obtainedwith the polynucleotide alone or the non-polynucleotide pesticidal agentalone. In an embodiment, a composition containing one or morepolynucleotides and one or more non-polynucleotide pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein, is found to effect synergistically improvedprevention or control of Leptinotarsa species infestations.

Controlling Leptinotarsa Infestations by Providing a DietaryPolynucleotide

Another aspect of this invention provides a method for controlling aLeptinotarsa species infestation of a plant comprising providing in thediet of a Leptinotarsa species an agent comprising a polynucleotidehaving at least one segment of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with a fragment ofequivalent length of a DNA having a sequence selected from the groupconsisting of: The Target Gene Sequences Group or the DNA complementthereof, wherein the agent functions upon ingestion by the Leptinotarsaspecies to inhibit a biological function within the Leptinotarsa speciesthereby controlling infestation by the Leptinotarsa species. Thepolynucleotide can be longer than the segment or segments it contains,but each polynucleotide segment and corresponding DNA fragment are ofequivalent length. Polynucleotides of use in the method can be designedfor multiple target genes. Embodiments include those in which the agentcomprises a dsRNA comprising a segment having a sequence selected fromthe group consisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114,or the complement thereof, or wherein the agent comprises apolynucleotide or RNA encoded by a sequence selected from the groupconsisting of SEQ ID NOs:1105-1109. In an embodiment, a method forcontrolling a Leptinotarsa species infestation of a plant comprisingproviding in the diet of the Leptinotarsa species a polynucleotidecomprising a nucleotide sequence that is complementary to at least 21contiguous nucleotides of a target gene having a nucleotide sequenceselected from the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094, or an RNA transcribed from thetarget gene is provided. In some embodiments, the polynucleotide is adouble-stranded RNA. In some embodiments, the polynucleotide (e.g.,double-stranded RNA) is chemically synthesized or is produced byexpression in a microorganism or by expression in a plant cell.Embodiments include those in which the polynucleotide is a dsRNA with astrand having a sequence selected from the group consisting of theTrigger Sequences Group. Related aspects of the invention includeisolated polynucleotides of use in the method and plants having improvedLeptinotarsa resistance provided by the method.

In various embodiments, the agent comprising a polynucleotide comprisesa microbial cell or is produced in a microorganism. For example, theagent can include or can be produced in bacteria or yeast cells. Inother embodiments the agent comprising a polynucleotide comprises atransgenic plant cell or is produced in a plant cell (for example aplant cell transiently expressing the polynucleotide); such plant cellscan be cells in an plant or cells grown in tissue culture or in cellsuspension.

In various embodiments, the agent comprising a polynucleotide isprovided for dietary uptake by the Leptinotarsa species in a formsuitable for ingestion, for example, as a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or as a seed treatment. The agent comprising apolynucleotide can be provided for dietary uptake by the Leptinotarsaspecies by applying the agent to a plant subject to infestation by theLeptinotarsa species or by applying the agent to seed of the plant, forexample by spraying, dusting, or coating the plant, or by application ofa soil drench, or by providing in an artificial diet. The agentcomprising a polynucleotide can be provided for dietary uptake by theLeptinotarsa species in an artificial diet formulated to meet theparticular nutritional requirements for maintaining the Leptinotarsaspecies, wherein the artificial diet is supplemented with some amount ofthe polynucleotide obtained from a separate source such as chemicalsynthesis or purified from a microbial fermentation; this embodiment canbe useful, e.g., for determining the timing and amounts of effectivepolynucleotide treatment regimes. In some embodiments the agentcomprising a polynucleotide is provided for dietary uptake by theLeptinotarsa species in the form of a plant cell or in plant cellcomponents, or in a microorganism (such as a bacterium or a yeast) or amicrobial fermentation product, or in a synthetic or man-made diet. Inone embodiment the agent comprising a polynucleotide is provided in theform of bait that is ingested by the Leptinotarsa species. The agentcomprising a polynucleotide can be provided for dietary uptake by theLeptinotarsa species in the form of a seed treatment, or in the form oftreatment of “seed potato” tubers or pieces of tuber (e.g., by soaking,coating, or dusting the seed potato). Suitable binders, inert carriers,surfactants, and the like can be included in the agent, as is known toone skilled in formulation of pesticides and seed treatments. In someembodiments, the agent comprising a polynucleotide further comprises oneor more components selected from the group consisting of a carrieragent, a surfactant, a cationic lipid (such as that disclosed in Example18 of U.S. patent application publication 2011/0296556, incorporated byreference herein), an organosilicone, an organosilicone surfactant, apolynucleotide herbicidal molecule, a non-polynucleotide herbicidalmolecule, a non-polynucleotide pesticide, a safener, and an insectgrowth regulator. In some embodiments, the agent comprising apolynucleotide further comprises at least one pesticidal agent selectedfrom the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein. In some embodiments, theagent comprising a polynucleotide comprises at least one implantableformulation selected from the group consisting of a particulate, pellet,or capsule implanted in the plant; in such embodiments the methodcomprises implanting in the plant the implantable formulation. In someembodiments, the agent comprising a polynucleotide comprises at leastone in-furrow formulation selected from the group consisting of apowder, granule, pellet, capsule, spray, or drench, or any other formssuited for applying to a furrow; in such embodiments, the methodcomprises an in-furrow treatment with the in-furrow formulation. In someembodiments, the method comprises treatment of a solanaceous plant seed,potato tuber, or piece of potato tuber with the agent.

It is anticipated that the combination of certain polynucleotides of usein agents of use in this method (e.g., the polynucleotide triggersdescribed in the working Examples) with one or more non-polynucleotidepesticidal agents will result in a synergetic improvement in preventionor control of Leptinotarsa species infestations, when compared to theeffect obtained with the polynucleotide alone or the non-polynucleotidepesticidal agent alone. In an embodiment, a composition containing oneor more polynucleotides and one or more non-polynucleotide pesticidalagent selected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein, is found to effect synergistically improvedprevention or control of Leptinotarsa species infestations when providedto the Leptinotarsa species in a diet.

In some embodiments, the polynucleotide is a dsRNA comprising a segmenthaving a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the polynucleotide is encoded by a sequence selected from thegroup consisting of SEQ ID NOs:1105-1109.

In some embodiments, the contiguous nucleotides have a sequence of about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identitywith a fragment of equivalent length of a DNA or target gene having asequence selected from The Target Gene Sequences Group or the DNAcomplement thereof. In some embodiments the contiguous nucleotides areexactly (100%) identical to a fragment of equivalent length of a DNA ortarget gene having a sequence selected from The Target Gene SequencesGroup or the DNA complement thereof. In some embodiments, thepolynucleotide has an overall sequence of about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identity with a fragment ofequivalent length of a DNA or target gene having a sequence selectedfrom The Target Gene Sequences Group or the DNA complement thereof. Inan embodiment, the polynucleotide comprises at least one segment of 21contiguous nucleotides with a sequence of 100% identity with thecorresponding fragment of a target gene having a DNA sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or the DNA complement thereof; in someembodiments, the polynucleotide comprises “neutral” sequence (having nosequence identity or complementarity to the target gene) in addition toa segment of 21 contiguous nucleotides with 100% identity with thecorresponding fragment of the target gene, and therefore thepolynucleotide as a whole is of much lower overall sequence identitywith a target gene.

The polynucleotide of use in this method is generally designed tosuppress one or more genes (“target genes”). The term “gene” refers toany portion of a nucleic acid that provides for expression of atranscript or encodes a transcript. A “gene” can include, but is notlimited to, a promoter region, 5′ untranslated regions, transcriptencoding regions that can include intronic regions, 3′ untranslatedregions, or combinations of these regions. In some embodiments, thetarget genes can include coding or non-coding sequence or both. In otherembodiments, the target gene has a sequence identical to orcomplementary to a messenger RNA, e.g., in some embodiments the targetgene is a cDNA. In specific embodiments, the polynucleotide is designedto suppress one or more target genes, where each target gene has a DNAsequence selected from the group consisting of the Target Gene SequencesGroup. In various embodiments, the polynucleotide is designed tosuppress one or more target genes, where each target gene has a sequenceselected from the group consisting of the Target Gene Sequences Group,and can be designed to suppress multiple target genes from this group,or to target different regions of one or more of these target genes. Inan embodiment, the polynucleotide comprises multiple segments of 21contiguous nucleotides with a sequence of 100% identity with a fragmentof equivalent length of a DNA or target gene having a sequence selectedfrom The Target Gene Sequences Group or the DNA complement thereof. Insuch cases, each segment can be identical or different in size or insequence, and can be sense or anti-sense relative to the target gene.For example, in one embodiment the polynucleotide comprises multiplesegments in tandem or repetitive arrangements, wherein each segmentcomprises 21 contiguous nucleotides with a sequence of 100% identitywith a fragment of equivalent length of a DNA or target gene having asequence selected from The Target Gene Sequences Group or the DNAcomplement thereof; the segments can be from different regions of thetarget gene, e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments.

The total length of the polynucleotide of use in this method can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with a fragment of equivalent length of a DNA ortarget gene having a sequence selected from The Target Gene SequencesGroup or the DNA complement thereof. In other words, the total length ofthe polynucleotide can be greater than the length of the section orsegment of the polynucleotide designed to suppress one or more targetgenes, where each target gene has a DNA sequence selected from the groupconsisting of the Target Gene Sequences Group. For example, thepolynucleotide can have nucleotides flanking the “active” segment of atleast one segment of 18 or more contiguous nucleotides that suppressesthe target gene, or include “spacer” nucleotides between activesegments, or can have additional nucleotides at the 5′ end, or at the 3′end, or at both the 5′ and 3′ ends. In an embodiment, the polynucleotidecan include additional nucleotides that are not specifically related(having a sequence not complementary or identical to) to the DNA ortarget gene having a sequence selected from The Target Gene SequencesGroup or the DNA complement thereof, e.g., nucleotides that providestabilizing secondary structure or for convenience in cloning ormanufacturing. In an embodiment, the polynucleotide can includeadditional nucleotides located immediately adjacent to one or moresegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with a fragment of equivalent length of a DNAor target gene having a sequence selected from The Target Gene SequencesGroup or the DNA complement thereof. In an embodiment, thepolynucleotide comprises one such segment, with an additional 5′ G or anadditional 3′ C or both, adjacent to the segment. In another embodiment,the polynucleotide is a double-stranded RNA comprising additionalnucleotides to form an overhang, for example, a dsRNA comprising 2deoxyribonucleotides to form a 3′ overhang. Thus in various embodiments,the nucleotide sequence of the entire polynucleotide is not 100%identical or complementary to a sequence of contiguous nucleotides inthe DNA or target gene having a sequence selected from The Target GeneSequences Group, or the DNA complement thereof. For example, in someembodiments the polynucleotide comprises at least two segments of 21contiguous nucleotides with a sequence of 100% identity with a fragmentof a DNA having a sequence selected from The Target Gene SequencesGroup, or the DNA complement thereof, wherein (1) the at least twosegments are separated by one or more spacer nucleotides, or (2) the atleast two segments are arranged in an order different from that in whichthe corresponding fragments occur in the DNA having a sequence selectedfrom The Target Gene Sequences Group, or the DNA complement thereof.

The polynucleotide of use in this method is provided by suitable meansknown to one in the art. Embodiments include those wherein thepolynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation.

In some embodiments the polynucleotide of use in this method is providedas an isolated DNA or RNA fragment. In some embodiments thepolynucleotide of use in this method is not part of an expressionconstruct and is lacking additional elements such as a promoter orterminator sequences. Such polynucleotides can be relatively short, suchas single- or double-stranded polynucleotides of between about 18 toabout 300 or between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or between about 18 to about 300 orbetween about 50 to about 500 base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. In some embodimentssuch recombinant expression constructs or vectors are designed toinclude additional elements, such as expression cassettes for expressinga gene of interest (e.g., an insecticidal protein).

Controlling Leptinotarsa Infestations by Providing a Dietary RNA

Another aspect of this invention provides a method of causing mortalityor stunting in larvae of the Leptinotarsa species by providing in thediet of the larvae at least one polynucleotide comprising at least onesilencing element comprising 21 contiguous nucleotides that arecomplementary to a target gene having a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or an RNA transcribed from the target gene. Insome embodiments, the polynucleotide is a double-stranded RNA. In someembodiments, the polynucleotide (e.g., double-stranded RNA) ischemically synthesized or is produced by expression in a microorganismor by expression in a plant cell. In an embodiment, a method of causingmortality or stunting in Leptinotarsa species larvae comprisingproviding in the diet of Leptinotarsa species larvae at least one RNAcomprising at least one silencing element essentially identical oressentially complementary to a fragment of a target gene sequence of theLeptinotarsa species larvae, wherein the target gene sequence isselected from the group consisting of the Target Gene Sequences Group,and wherein ingestion of the RNA by the Leptinotarsa species larvaeresults in mortality or stunting in the Leptinotarsa species larvae isprovided. A related aspect of this invention is an RNA comprising atleast one silencing element, wherein the at least one silencing elementis essentially identical or essentially complementary to a fragment of atarget gene of the Leptinotarsa species larvae, wherein the target genesequence is selected from the group consisting of the Target GeneSequences Group. The RNA can be longer than the silencing element orsilencing elements it contains, but each silencing element andcorresponding fragment of a target gene sequence are of equivalentlength. RNAs of use in the method can be designed for multiple targetgenes; embodiments include RNAs comprising at least one silencingelement comprising a sequence selected from the group consisting of: SEQID NOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the silencing element is encoded by a sequence selected from thegroup consisting of SEQ ID NOs:1105-1109. Embodiments include those inwhich the RNA comprises a dsRNA with a strand having a sequence selectedfrom the group consisting of the Trigger Sequences Group. In a relatedaspect, a method of causing mortality or lower fecundity in Leptinotarsaspecies comprising providing in the diet of Leptinotarsa species atleast one RNA comprising at least one silencing element essentiallyidentical or essentially complementary to a fragment of a target genesequence of the Leptinotarsa species larvae, wherein the target genesequence is selected from The Target Gene Sequences Group, or the DNAcomplement thereof, and wherein ingestion of the RNA by the Leptinotarsaspecies results in mortality or lower fecundity in the Leptinotarsaspecies is provided. Related aspects of the invention include isolatedRNAs of use in the method and plants having improved Leptinotarsaresistance provided by the method.

In various embodiments, the diet providing the RNA comprises a microbialcell or is produced in a microorganism. For example, the diet providingthe RNA can include or can be produced in bacteria or yeast cells. Insimilar embodiments the diet providing the RNA comprises a transgenicplant cell or is produced in a plant cell (for example a plant celltransiently expressing the polynucleotide); such plant cells can becells in an plant or cells grown in tissue culture or in cellsuspension.

In one embodiment the diet providing the RNA is provided in the form ofany plant that is subject to infestation by a Leptinotarsa species,wherein the RNA is contained in or on the plant. Such plants can bestably transgenic plants that express the RNA, or non-transgenic plantsthat transiently express the RNA or that have been treated with the RNA,e.g., by spraying or coating. Stably transgenic plants generally containintegrated into their genome a recombinant construct that encodes theRNA. Of particular interest are embodiments wherein the plant is asolanaceous plant (family Solanaceae). Examples include a plant selectedfrom the group consisting of potato, tomato, and eggplant. Embodimentsinclude those wherein the plant is an ungerminated solanaceous plantseed, a solanaceous plant in a vegetative stage, or a solanaceous plantin a reproductive stage. Embodiments include those wherein the plant isa “seed potato”, meaning a potato tuber or piece of potato tuber whichcan be propagated into new potato plants.

In various embodiments, the diet providing the RNA is provided in a formsuitable for ingestion by the Leptinotarsa species, for example, as asolid, liquid (including homogeneous mixtures such as solutions andnon-homogeneous mixtures such as suspensions, colloids, micelles, andemulsions), powder, suspension, emulsion, spray, encapsulated ormicro-encapsulation formulation, in or on microbeads or other carrierparticulates, in a film or coating, or on or within a matrix, or as aseed treatment. The diet providing the RNA can be provided by applyingthe diet to a plant subject to infestation by the Leptinotarsa species,for example by spraying, dusting, or coating the plant, or byapplication of a soil drench, or by providing in an artificial diet. Inone embodiment the diet providing the recombinant RNA is provided in theform of bait that is ingested by the Leptinotarsa species. The dietproviding the RNA can be an artificial diet formulated to meet theparticular nutritional requirements for maintaining the Leptinotarsaspecies, wherein the artificial diet is supplemented with some amount ofthe RNA obtained from a separate source such as chemical synthesis orpurified from a microbial fermentation; this embodiment can be useful,e.g., for determining the timing and amounts of effective polynucleotidetreatment regimes. In some embodiments the diet providing the RNA isprovided in the form of a plant cell or in plant cell components, or ina microorganism (such as a bacterium or a yeast) or a microbialfermentation product, or in a synthetic diet. In one embodiment the dietproviding the RNA is provided in the form of bait that is ingested bythe Leptinotarsa species. The diet providing the RNA can be provided inthe form of a seed treatment, or in the form of treatment of “seedpotato” tubers or pieces of tuber (e.g., by soaking, coating, or dustingthe seed potato). Suitable binders, inert carriers, surfactants, and thelike can be included in the diet, as is known to one skilled informulation of pesticides and seed treatments. In some embodiments, thediet providing the RNA further comprises one or more components selectedfrom the group consisting of a carrier agent, a surfactant, a cationiclipid (such as that disclosed in Example 18 of U.S. patent applicationpublication 2011/0296556, incorporated by reference herein), anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator.In some embodiments, the diet providing the RNA further comprises atleast one pesticidal agent selected from the group consisting of apatatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein. In some embodiments, thediet providing the RNA includes at least one implantable formulationselected from the group consisting of a particulate, pellet, or capsuleimplanted in the plant; in such embodiments the method comprisesimplanting in the plant the implantable formulation. In someembodiments, the diet providing the RNA includes at least one in-furrowformulation selected from the group consisting of a powder, granule,pellet, capsule, spray, or drench, or any other forms suited forapplying to a furrow; in such embodiments, the method includes anin-furrow treatment with the in-furrow formulation. In some embodiments,the method comprises treatment of a solanaceous plant seed, potatotuber, or piece of potato tuber with the agent.

It is anticipated that the combination of certain RNAs of use in thismethod (e.g., the dsRNA triggers described in the working Examples) withone or more non-polynucleotide pesticidal agents will result in asynergetic improvement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with the RNA alone orthe non-polynucleotide pesticidal agent alone. In an embodiment, acomposition containing one or more RNAs and one or morenon-polynucleotide pesticidal agent selected from the group consistingof a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein, isfound to effect synergistically improved prevention or control ofLeptinotarsa species infestations.

The RNA of use in this method can be single-stranded (ss) ordouble-stranded (ds). Embodiments of the method include those whereinthe RNA is at least one selected from the group consisting of sensesingle-stranded RNA (ssRNA), anti-sense single-stranded (ssRNA), ordouble-stranded RNA (dsRNA); a mixture of RNAs of any of these types canbe used. In one embodiment a double-stranded DNA/RNA hybrid is used. TheRNA can include components other than standard ribonucleotides, e.g., anembodiment is an RNA that comprises terminal deoxyribonucleotides.

The RNA comprises at least one silencing element, wherein the silencingelement is essentially identical (as the RNA equivalent) or essentiallycomplementary to a fragment of a target gene of the Leptinotarsa specieslarvae, wherein the target gene sequence is selected from the groupconsisting of the Target Gene Sequences Group. In some embodiments, thesilencing element has a sequence of about 95%, about 96%, about 97%,about 98%, about 99%, or about 100% identity with or complementarity toa fragment of equivalent length of a DNA having a sequence selected fromthe group consisting of the Target Gene Sequences Group. In someembodiments the silencing element is exactly (100%) identical or exactly(100%) complementary (as the RNA equivalent) to a fragment of equivalentlength of a DNA having a sequence selected from The Target GeneSequences Group or the DNA complement thereof. In some embodiments, theRNA containing the silencing element(s) has an overall sequence of about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identitywith or complementarity to the fragment of a DNA having a sequenceselected from the group consisting of the Target Gene Sequences Group.

In some embodiments, the silencing element comprises at least onesegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with or complementarity to a fragment ofequivalent length of the target gene. In some embodiments the silencingelement comprises at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with orcomplementarity to a fragment of equivalent length of a DNA having asequence selected from the group consisting of the Target Gene SequencesGroup. In some embodiments the silencing element comprises at least onesegment of 18 or more contiguous nucleotides, e.g., between 18-24, orbetween 18-28, or between 20-30, or between 20-50, or between 20-100, orbetween 50-100, or between 50-500, or between 100-250, or between100-500, or between 200-1000, or between 500-2000, or even greater. Insome embodiments the silencing element comprises more than 18 contiguousnucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, orgreater than 30, e.g., about 35, about 40, about 45, about 50, about 55,about 60, about 65, about 70, about 75, about 80, about 85, about 90,about 95, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170, about 180, about 190, about 200, about 210,about 220, about 230, about 240, about 250, about 260, about 270, about280, about 290, about 300, about 350, about 400, about 450, about 500,or greater than 500 contiguous nucleotides. In particular embodiments,the silencing element comprises at least one segment of at least 21contiguous nucleotides with a sequence of 100% identity with a fragmentof equivalent length of a DNA or target gene having a sequence selectedfrom The Target Gene Sequences Group or the DNA complement thereof. Inparticular embodiments, the RNA is a double-stranded nucleic acid (e.g.,dsRNA) with one strand comprising at least one segment of at least 21contiguous nucleotides with a sequence of 100% identity with a fragmentof equivalent length of a DNA or target gene having a sequence selectedfrom The Target Gene Sequences Group or the DNA complement thereof;expressed as base-pairs, such a double-stranded nucleic acid comprisesat least one segment of at least 21 contiguous, perfectly matchedbase-pairs which correspond to a fragment of equivalent length of a DNAor target gene having a sequence selected from The Target Gene SequencesGroup or the DNA complement thereof. In particular embodiments, eachsilencing element contained in the RNA is of a length greater than thatwhich is typical of naturally occurring regulatory small RNAs, e.g.,each segment is at least about 30 contiguous nucleotides (or base-pairs)in length. In some embodiments, the total length of the RNA, or thelength of each silencing element contained in the RNA, is less than thetotal length of the sequence of interest (DNA or target gene having asequence selected from the group consisting of the Target Gene SequencesGroup). In some embodiments, the total length of the RNA is betweenabout 50 to about 500 nucleotides (for single-stranded polynucleotides)or base-pairs (for double-stranded polynucleotides). In someembodiments, the RNA is a dsRNA of between about 100 to about 500base-pairs, such as a dsRNA of the length of any of the dsRNA triggersdisclosed in Tables 3, 5, 8, 9, and 10. Embodiments include those inwhich the RNA is a dsRNA comprising a segment having a sequence selectedfrom the group consisting of: SEQ ID NOs:831-1085, 1095-1104, and1110-1114, or the complement thereof, or wherein the RNA is encoded by asequence selected from the group consisting of SEQ ID NOs:1105-1109.

The RNA of use in this method is generally designed to suppress one ormore genes (“target genes”). The term “gene” refers to any portion of anucleic acid that provides for expression of a transcript or encodes atranscript. A “gene” can include, but is not limited to, a promoterregion, 5′ untranslated regions, transcript encoding regions that caninclude intronic regions, 3′ untranslated regions, or combinations ofthese regions. In some embodiments, the target genes can include codingor non-coding sequence or both. In other embodiments, the target genehas a sequence identical to or complementary to a messenger RNA, e.g.,in some embodiments the target gene is a cDNA. In specific embodiments,the RNA is designed to suppress one or more target genes, where eachtarget gene has a DNA sequence selected from the group consisting of theTarget Gene Sequences Group. In various embodiments, the RNA is designedto suppress one or more genes, where each gene has a sequence selectedfrom the group consisting of the Target Gene Sequences Group, and can bedesigned to suppress multiple genes from this group, or to targetdifferent regions of one or more of these genes. In an embodiment, theRNA comprises multiple silencing elements each of which comprises atleast one segment of 21 contiguous nucleotides with a sequence of 100%identity with or 100% complementarity to a fragment of equivalent lengthof a DNA having a sequence selected from The Target Gene Sequences Groupor the DNA complement thereof. In such cases, each silencing element canbe identical or different in size or in sequence, and can be sense oranti-sense relative to the target gene. For example, in one embodimentthe RNA can include multiple silencing elements in tandem or repetitivearrangements, wherein each silencing element comprises at least onesegment of 21 contiguous nucleotides with a sequence of 100% identitywith or 100% complementarity to a fragment of equivalent length of a DNAhaving a sequence selected from the group consisting of the Target GeneSequences Group; the segments can be from different regions of thetarget gene, e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments.

The total length of the RNA can be greater than 18 contiguousnucleotides, and can include nucleotides in addition to the silencingelement having a sequence of about 95% to about 100% identity with orcomplementarity to a fragment of equivalent length of a DNA or targetgene having a sequence selected from the group consisting of the TargetGene Sequences Group. In other words, the total length of the RNA can begreater than the length of the silencing element designed to suppressone or more target genes, where each target gene has a DNA sequenceselected from the group consisting of the Target Gene Sequences Group.For example, the RNA can have nucleotides flanking the “active”silencing element of at least one segment of 18 or more contiguousnucleotides that suppresses the target gene, or include “spacer”nucleotides between active silencing elements, or can have additionalnucleotides at the 5′ end, or at the 3′ end, or at both the 5′ and 3′ends. In an embodiment, the RNA comprises additional nucleotides thatare not specifically related (ihaving a sequence not complementary oridentical to) to the DNA or target gene having a sequence selected fromThe Target Gene Sequences Group or the DNA complement thereof, e.g.,nucleotides that provide stabilizing secondary structure or forconvenience in cloning or manufacturing. In an embodiment, the RNAcomprises additional nucleotides located immediately adjacent to one ormore silencing element of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with or complementarity toa fragment of equivalent length of a DNA or target gene having asequence selected from the group consisting of the Target Gene SequencesGroup. In an embodiment, the RNA comprises one such silencing element,with an additional 5′ G or an additional 3′ C or both, adjacent to thesilencing element. In another embodiment, the RNA is a double-strandedRNA comprising additional nucleotides to form an overhang, for example,a dsRNA comprising 2 deoxyribonucleotides to form a 3′ overhang. Thus invarious embodiments, the nucleotide sequence of the entire RNA is not100% identical or complementary to a fragment of contiguous nucleotidesin the DNA or target gene having a sequence selected from the groupconsisting of the Target Gene Sequences Group. For example, in someembodiments the RNA comprises at least two silencing elements each of 21contiguous nucleotides with a sequence of 100% identity with a fragmentof a DNA having a sequence selected from the group consisting of theTarget Gene Sequences Group4, or the DNA complement thereof, wherein (1)the at least two silencing elements are separated by one or more spacernucleotides, or (2) the at least two silencing elements are arranged inan order different from that in which the corresponding fragments occurin the DNA having a sequence selected from the group consisting of theTarget Gene Sequences Group, or the DNA complement thereof.

In some embodiments the RNA consists of naturally occurringribonucleotides. In certain embodiments, the RNA comprises componentsother than ribonucleotides, for example, synthetic RNAs consistingmainly of ribonucleotides but with one or more terminaldeoxyribonucleotides or one or more terminal dideoxyribonucleotides. Incertain embodiments, the RNA comprises non-canonical nucleotides such asinosine, thiouridine, or pseudouridine. In certain embodiments, the RNAcomprises chemically modified nucleotides.

The RNA of use in this method is provided by suitable means known to onein the art. Embodiments include those wherein the RNA is chemicallysynthesized (e.g., by in vitro transcription, such as transcriptionusing a T7 polymerase or other polymerase), produced by expression in amicroorganism or in cell culture (such as plant or insect cells grown inculture), produced by expression in a plant cell, or produced bymicrobial fermentation.

In some embodiments the RNA is provided as an isolated RNA that is notpart of an expression construct and is lacking additional elements suchas a promoter or terminator sequences. Such RNAs can be relativelyshort, such as single- or double-stranded RNAs of between about 18 toabout 300 or between about 50 to about 500 nucleotides (forsingle-stranded RNAs) or between about 18 to about 300 or between about50 to about 500 base-pairs (for double-stranded RNAs). Alternatively theRNA can be provided in more complex constructs, e.g., as part of arecombinant expression construct, or included in a recombinant vector,for example in a recombinant plant virus vector or in a recombinantbaculovirus vector. In some embodiments such recombinant expressionconstructs or vectors are designed to include additional elements, suchas including additional RNA encoding an aptamer or ribozyme or anexpression cassette for expressing a gene of interest (e.g., aninsecticidal protein).

Methods of Providing Plants Having Improved Resistance to LeptinotarsaInfestations, and the Plants, Plant Parts, and Seeds Thus Provided

Another aspect of this invention provides a method of providing a planthaving improved resistance to a Leptinotarsa species infestationcomprising topically applying to the plant a composition comprising atleast one polynucleotide having at least one segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity with a fragment of a target gene or DNA having a sequenceselected from The Target Gene Sequences Group, or the DNA complementthereof, in a manner such that the plant treated with thepolynucleotide-containing composition exhibits improved resistance to aLeptinotarsa species infestation, relative to an untreated plant. In anembodiment, the at least one polynucleotide comprises at least onesegment of 18 or more contiguous nucleotides that are essentiallyidentical to a fragment of equivalent length of a DNA having a sequenceselected from the Target Gene Sequences Group, or the DNA complementthereof. The polynucleotide can be longer than the segment or segmentsit contains, but each segment and corresponding fragment of a targetgene are of equivalent length. In an embodiment, this invention providesa method of providing a plant having improved resistance to aLeptinotarsa species infestation comprising topically applying to theplant a composition comprising at least one polynucleotide comprising anucleotide sequence that is complementary to at least 21 contiguousnucleotides of a target gene having a nucleotide sequence selected fromthe group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725,SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or an RNA transcribed from the target gene. In anembodiment, this invention provides a method of providing a plant havingimproved resistance to a Leptinotarsa species infestation comprisingtopically applying to the plant a composition comprising at least onepolynucleotide in a manner such that an effective amount of thepolynucleotide is ingested by Leptinotarsa species feeding on the plant,the polynucleotide comprising at least 21 contiguous nucleotides thatare complementary to a target gene having a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or an RNA transcribed from the target gene. Insome embodiments, this invention provides a method for controlling aLeptinotarsa species infestation of a plant comprising topicallyapplying to the plant a composition comprising at least onepolynucleotide in a manner such that an effective amount of thepolynucleotide is ingested by Leptinotarsa species feeding on the plant,the polynucleotide comprising a nucleotide sequence that iscomplementary to at least 21 contiguous nucleotides of a target genehaving a nucleotide sequence selected from the group consisting of: SEQID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ ID NOs:726-729, SEQ IDNOs:731-806, SEQ ID NOs:808-830, and SEQ ID NOs:1087-1094, or an RNAtranscribed from the target gene; wherein the Leptinotarsa species isLeptinotarsa decemlineata; and wherein the target gene has the sequenceof SEQ ID NO:730 or wherein the polynucleotide is a double-stranded RNAhaving a strand with a sequence selected from the group consisting ofSEQ ID NO:989, 988, 1104, or 1105. Polynucleotides of use in the methodcan be designed for multiple target genes. Embodiments include those inwhich the polynucleotide comprises a segment having a sequence selectedfrom the group consisting of: SEQ ID NOs:831-1085, 1095-1104, and1110-1114, or the complement thereof, or wherein the polynucleotide isencoded by a sequence selected from the group consisting of SEQ IDNOs:1105-1109. Embodiments include those in which the compositioncomprises a dsRNA with a strand having a sequence selected from thegroup consisting of the Trigger Sequences Group. Related aspects of theinvention include compositions for topical application and isolatedpolynucleotides of use in the method, and plants having improvedLeptinotarsa resistance provided by the method.

By “topical application” is meant application to the surface or exteriorof an object, such as the surface or exterior of a plant, such asapplication to the surfaces of a plant part such as a leaf, stem,flower, fruit, shoot, root, seed, tuber, flowers, anthers, or pollen, orapplication to an entire plant, or to the above-ground or below-groundportions of a plant. Topical application can be carried out onnon-living surfaces, such as application to soil, or to a surface ormatrix by which a Leptinotarsa insect can come in contact with thepolynucleotide. In various embodiments of the method, the compositioncomprising at least one polynucleotide is topically applied to the plantin a suitable form, e.g., as a solid, liquid (including homogeneousmixtures such as solutions and non-homogeneous mixtures such assuspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or as a seed treatment. In some embodiments of themethod, the polynucleotide-containing composition is topically appliedto above-ground parts of the plant, e.g., sprayed or dusted onto leaves,stems, and flowering parts of the plant. Embodiments of the methodinclude topical application of a foliar spray (e.g., spraying a liquidpolynucleotide-containing composition on leaves of a solanaceous plant)or a foliar dust (e.g., dusting a solanaceous plant with apolynucleotide-containing composition in the form of a powder or oncarrier particulates). In other embodiments, thepolynucleotide-containing composition is topically applied tobelow-ground parts of the plant, such as to the roots, e.g., by means ofa soil drench. In other embodiments, the polynucleotide-containingcomposition is topically applied to a seed that is grown into the plant.The topical application can be in the form of topical treatment offruits of solanaceous plants or seeds from fruits of solanaceous plants,or in the form of topical treatment of “seed potato” tubers or pieces oftuber (e.g., by soaking, coating, or dusting the seed potato). Suitablebinders, inert carriers, surfactants, and the like can optionally beincluded in the polynucleotide-containing composition, as is known toone skilled in formulation of pesticides and seed treatments. In someembodiments, the polynucleotide-containing composition is at least onetopically implantable formulation selected from the group consisting ofa particulate, pellet, or capsule topically implanted in the plant; insuch embodiments the method comprises topically implanting in the plantthe topically implantable formulation. In some embodiments, thepolynucleotide-containing composition is at least one in-furrowformulation selected from the group consisting of a powder, granule,pellet, capsule, spray, or drench, or any other forms suited fortopically applying to a furrow; in such embodiments, the method includesan in-furrow treatment with the in-furrow formulation. In one embodimentthe polynucleotide-containing composition can be ingested or otherwiseabsorbed internally by the Leptinotarsa species. For example, thepolynucleotide-containing composition can be in the form of bait. Insome embodiments, the polynucleotide-containing composition furthercomprises one or more components selected from the group consisting of acarrier agent, a surfactant, a cationic lipid (such as that disclosed inExample 18 of U.S. patent application publication 2011/0296556,incorporated by reference herein), an organosilicone, an organosiliconesurfactant, a polynucleotide herbicidal molecule, a non-polynucleotideherbicidal molecule, a non-polynucleotide pesticide, a safener, and aninsect growth regulator. In one embodiment the composition furthercomprises a nonionic organosilicone surfactant such as SILWET® brandsurfactants, e.g., SILWET L-77® brand surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, currentlyavailable from Momentive Performance Materials, Albany, N.Y. In someembodiments, the topically applied composition further comprises atleast one pesticidal agent selected from the group consisting of apatatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein. Alternatively suchadditional components or pesticidal agents can be provided separately,e.g., by separate topical application or by transgenic expression in theplant. Alternatively the plant is topically treated with thepolynucleotide-containing composition as well as with a separate(preceding, following, or concurrent) application of a substance thatimproves the efficacy of the polynucleotide-containing composition. Forexample, a plant can be sprayed with a first topical application of asolution containing a nonionic organosilicone surfactant such as SILWET®brand surfactants, e.g., SILWET L-77® brand surfactant, followed by asecond topical application of the polynucleotide-containing composition,or vice-versa.

It is anticipated that the combination of certain polynucleotides usefulin the polynucleotide-containing composition (e.g., the polynucleotidetriggers described in the working Examples) with one or morenon-polynucleotide pesticidal agents will result in a synergeticimprovement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with thepolynucleotide alone or the non-polynucleotide pesticidal agent alone.In an embodiment, the polynucleotide-containing composition is providedas a transgenic plant expressing one or more polynucleotides and one ormore genes encoding a non-polynucleotide pesticidal agent selected fromthe group consisting of a patatin, a plant lectin, a phytoecdysteroid, aBacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidalprotein, a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein,wherein the transgenic plant is found to exhibit synergisticallyimproved resistance to Leptinotarsa species infestations.

The polynucleotide useful in the polynucleotide-containing compositionis provided by suitable means known to one in the art. Embodimentsinclude those wherein the polynucleotide is chemically synthesized(e.g., by in vitro transcription, such as transcription using a T7polymerase or other polymerase), produced by expression in amicroorganism or in cell culture (such as plant or insect cells grown inculture), produced by expression in a plant cell, or produced bymicrobial fermentation.

In many embodiments the polynucleotide useful in thepolynucleotide-containing composition is provided as an isolated DNA orRNA fragment. In some embodiments the polynucleotide useful in thepolynucleotide-containing composition is not part of an expressionconstruct and is lacking additional elements such as a promoter orterminator sequences). Such polynucleotides can be relatively short,such as single- or double-stranded polynucleotides of between about 18to about 300 or between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or between about 18 to about 300 orbetween about 50 to about 500 base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. Such recombinantexpression constructs or vectors can be designed to include additionalelements, such as expression cassettes for expressing a gene of interest(e.g., an insecticidal protein).

The polynucleotide useful in the polynucleotide-containing compositionhas at least one segment of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In an embodiment thepolynucleotide comprises at least one segment of 18 or more contiguousnucleotides that are essentially identical or complementary to afragment of equivalent length of a DNA having a sequence selected fromthe group consisting of the Target Gene Sequences Group. In someembodiments, the contiguous nucleotides have a sequence of about 95%,about 96%, about 97%, about 98%, about 99%, or about 100% identity witha fragment of a DNA having a sequence selected from the group consistingof: SEQ ID NOs:1-725 or SEQ ID NOs:726-830 or SEQ ID NOs:1087-1094 orthe DNA complement thereof. In some embodiments the contiguousnucleotides are exactly (100%) identical to a fragment of equivalentlength of a DNA having a sequence selected from the Target GeneSequences Group or the DNA complement thereof. In some embodiments, thepolynucleotide has an overall sequence of about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identity with a fragment of aDNA having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof.

The polynucleotide useful in the polynucleotide-containing compositioncomprises at least one segment of 18 or more contiguous nucleotides witha sequence of about 95% to about 100% identity with a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In some embodimentsthe polynucleotide comprises at least one segment of 18 or morecontiguous nucleotides, e.g., between 18-24, or between 18-28, orbetween 20-30, or between 20-50, or between 20-100, or between 50-100,or between 50-500, or between 100-250, or between 100-500, or between200-1000, or between 500-2000, or even greater. In some embodiments thesegment comprises more than 18 contiguous nucleotides, e.g., 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., about 35,about 40, about 45, about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 110,about 120, about 130, about 140, about 150, about 160, about 170, about180, about 190, about 200, about 210, about 220, about 230, about 240,about 250, about 260, about 270, about 280, about 290, about 300, about350, about 400, about 450, about 500, or greater than 500 contiguousnucleotides. In particular embodiments, the polynucleotide comprises atleast one segment of at least 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of equivalent length of a DNA or targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof. In particular embodiments, thepolynucleotide is a double-stranded nucleic acid (e.g., dsRNA) with onestrand comprising at least one segment of at least 21 contiguousnucleotides with a sequence of 100% identity with a fragment ofequivalent length of a DNA or target gene having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereofexpressed as base-pairs, such a double-stranded nucleic acid comprisesat least one segment of at least 21 contiguous, perfectly matchedbase-pairs which correspond to a fragment of equivalent length of a DNAor target gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof. In particular embodiments, eachsegment contained in the polynucleotide is of a length greater than thatwhich is typical of naturally occurring regulatory small RNAs, e.g.,each segment is at least about 30 contiguous nucleotides (or base-pairs)in length. In some embodiments, the total length of the polynucleotide,or the length of each segment contained in the polynucleotide, is lessthan the total length of the sequence of interest (DNA or target genehaving a sequence selected from the group consisting of the Target GeneSequences Group). In some embodiments, the total length of thepolynucleotide is between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10. Insome embodiments, the polynucleotide is a dsRNA comprising a segmenthaving a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orthe polynucleotide is encoded by a sequence selected from the groupconsisting of SEQ ID NOs:1105-1109.

The topically applied polynucleotide is generally designed to suppressone or more genes (“target genes”). Such target genes can include codingor non-coding sequence or both. In specific embodiments, thepolynucleotide is designed to suppress one or more target genes, whereeach target gene has a DNA sequence selected from the group consistingof the Target Gene Sequences Group. In various embodiments, thetopically applied polynucleotide is designed to suppress one or moregenes, where each gene has a sequence selected from the group consistingof the Target Gene Sequences Group, and can be designed to suppressmultiple genes from this group, or to target different regions of one ormore of these genes. In an embodiment, the topically appliedpolynucleotide comprises multiple sections or segments each of whichcomprises at least one segment of 21 contiguous nucleotides with asequence of 100% identity with a fragment of equivalent length of a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof. In such cases, each section can be identical ordifferent in size or in sequence, and can be sense or anti-senserelative to the target gene. For example, in one embodiment thetopically applied polynucleotide can include multiple sections in tandemor repetitive arrangements, wherein each section comprises at least onesegment of 21 contiguous nucleotides with a sequence of 100% identitywith a fragment of equivalent length of a DNA having a sequence selectedfrom the group consisting of: SEQ ID NOs:1-725 or SEQ ID NOs:726-830 orSEQ ID NOs:1087-1094 or the DNA complement thereof the segments can befrom different regions of the target gene, e.g., the segments cancorrespond to different exon regions of the target gene, and “spacer”nucleotides which do not correspond to a target gene can optionally beused in between or adjacent to the segments.

The total length of the topically applied polynucleotide can be greaterthan 18 contiguous nucleotides, and can include nucleotides in additionto the contiguous nucleotides having the sequence of about 95% to about100% identity with a fragment of equivalent length of a DNA having asequence selected from the Target Gene Sequences Group or the DNAcomplement thereof. In other words, the total length of the topicallyapplied polynucleotide can be greater than the length of the section orsegment of the polynucleotide designed to suppress one or more targetgenes, where each target gene has a DNA sequence selected from the groupconsisting of the Target Gene Sequences Group. For example, thetopically applied polynucleotide can have nucleotides flanking the“active” segment of at least one segment of 18 or more contiguousnucleotides that suppresses the target gene, or include “spacer”nucleotides between active segments, or can have additional nucleotidesat the 5′ end, or at the 3′ end, or at both the 5′ and 3′ ends. In anembodiment, the topically applied polynucleotide comprises additionalnucleotides that are not specifically related (having a sequence notcomplementary or identical to) to the DNA or target gene having asequence selected from the Target Gene Sequences Group or the DNAcomplement thereof, e.g., nucleotides that provide stabilizing secondarystructure or for convenience in cloning or manufacturing. In anembodiment, the topically applied polynucleotide comprises additionalnucleotides located immediately adjacent to one or more segment of 18 ormore contiguous nucleotides with a sequence of about 95% to about 100%identity with or complementarity to a fragment of equivalent length of aDNA or target gene having a sequence selected from the group consistingof the Target Gene Sequences Group. In an embodiment, the topicallyapplied polynucleotide comprises one such segment, with an additional 5′G or an additional 3′ C or both, adjacent to the segment. In anotherembodiment, the topically applied polynucleotide is a double-strandedRNA comprising additional nucleotides to form an overhang, for example,a dsRNA comprising 2 deoxyribonucleotides to form a 3′ overhang. Thus invarious embodiments, the nucleotide sequence of the entire topicallyapplied polynucleotide is not 100% identical or complementary to afragment of contiguous nucleotides in the DNA or target gene having asequence selected from the group consisting of the Target Gene SequencesGroup. For example, in some embodiments the topically appliedpolynucleotide comprises at least two segments each of 21 contiguousnucleotides with a sequence of 100% identity with a fragment of a DNAhaving a sequence selected from the Target Gene Sequences Group, or theDNA complement thereof, wherein (1) the at least two segments areseparated by one or more spacer nucleotides, or (2) the at least twosegments are arranged in an order different from that in which thecorresponding fragments occur in the DNA having a sequence selected fromthe Target Gene Sequences Group, or the DNA complement thereof.

In a related aspect, this invention is directed to the plant havingimproved resistance to a Leptinotarsa species infestation, provided bythis method which comprises topically applying to the plant acomposition comprising at least one polynucleotide having at least onesegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with a fragment of equivalent length of a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof, whereby the plant treated with thepolynucleotide composition exhibits improved resistance to aLeptinotarsa species infestation, relative to an untreated plant. Anembodiment is a solanaceous plant having improved resistance to aLeptinotarsa species infestation when compared to a control plant,provided by topically applying to the plant or to a seed grown into theplant (or, where the plant is a potato plant, to a seed potato growninto the potato plant) a dsRNA trigger having a sequence selected fromthe group consisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114,or the complement thereof, or a dsRNA trigger encoded by a sequenceselected from the group consisting of SEQ ID NOs:1105-1109. In yetanother aspect, this invention is directed to seed (especiallytransgenic progeny seed) produced by the plant having improvedresistance to a Leptinotarsa species infestation, as provided by thismethod. Also contemplated is a commodity product produced by the planthaving improved resistance to a Leptinotarsa species infestation, asprovided by this method, and a commodity product produced from thetransgenic progeny seed of such a plant.

Insecticidal Compositions for Controlling Leptinotarsa Species

Another aspect of this invention provides an insecticidal compositionfor controlling a Leptinotarsa species comprising an insecticidallyeffective amount of at least one RNA comprising at least one segment of18 or more contiguous nucleotides that is essentially identical orcomplementary to a fragment of a target gene or DNA having a sequenceselected from the group consisting of the Target Gene Sequences Group.In this context “controlling” includes inducement of a physiological orbehavioural change in a Leptinotarsa species (adult or larvae) such as,but not limited to, growth stunting, increased mortality, decrease inreproductive capacity, decrease in or cessation of feeding behavior ormovement, or decrease in or cessation of metamorphosis stagedevelopment. By “insecticidally effective” is meant effective ininducing a physiological or behavioural change in a Leptinotarsa species(adult or larvae) such as, but not limited to, growth stunting,increased mortality, decrease in reproductive capacity or decreasedfecundity, decrease in or cessation of feeding behavior or movement, ordecrease in or cessation of metamorphosis stage development; in someembodiments, application of an insecticidally effective amount of theRNA to a plant improves the plant's resistance to infestation by aLeptinotarsa species. The RNA can be longer than the segment or segmentsit contains, but each segment and corresponding fragment of a targetgene are of equivalent length. RNAs of use in the method can be designedfor multiple target genes. Embodiments include those in which theinsecticidal composition comprises an insecticidally effective amount ofa polynucleotide comprising at least 21 contiguous nucleotides that arecomplementary to a target gene having a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or an RNA transcribed from the target gene; oran insecticidally effective amount of at least one polynucleotidecomprising at least one silencing element that is complementary to atleast 21 contiguous nucleotides of a target gene or an RNA transcribedfrom the target gene, wherein the target gene has a nucleotide sequenceselected from the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094; or an insecticidally effectiveamount of at least one RNA comprising at least one segment that isidentical or complementary to at least 21 contiguous nucleotides of atarget gene having a nucleotide sequence selected from the groupconsisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQ IDNOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or an RNA transcribed from the target gene; or an RNAmolecule that causes mortality or stunting of growth in a Leptinotarsaspecies when ingested or contacted by the Leptinotarsa species, whereinthe RNA molecule comprises at least 21 contiguous nucleotides that arecomplementary to a target gene having a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ IDNOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830,and SEQ ID NOs:1087-1094, or an RNA transcribed from the target gene; oran insecticidal double-stranded RNA molecule that causes mortality orstunting of growth in a Leptinotarsa species when ingested or contactedby the Leptinotarsa species, wherein at least one strand of theinsecticidal double-stranded RNA molecule comprises 21 contiguousnucleotides that are complementary to a target gene or an RNAtranscribed from the target gene, wherein the target gene has a sequenceselected from the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094; or an insecticidally effectiveamount of at least one double-stranded RNA comprising a sequenceselected from the Trigger Sequences Group. In some embodiments, thepolynucleotide is a double-stranded RNA. In some embodiments, thepolynucleotide (e.g., double-stranded RNA) is chemically synthesized oris produced by expression in a microorganism or by expression in a plantcell. Embodiments include insecticidal compositions comprising a dsRNAhaving a sequence selected from the group consisting of SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the insecticidal composition comprises a polynucleotide or RNAencoded by a sequence selected from the group consisting of SEQ IDNOs:1105-1109. Embodiments include those in which the insecticidalcomposition comprises a dsRNA with a strand having a sequence selectedfrom the group consisting of the Trigger Sequences Group. In anembodiment this invention provides an insecticidal composition forcontrolling a Leptinotarsa species comprising an insecticidallyeffective amount of a double-stranded RNA molecule that causes mortalityor stunting of growth in a Leptinotarsa species when ingested orcontacted by the Leptinotarsa species, wherein the insecticidaldouble-stranded RNA molecule comprises at least one segment that iscomplementary to 21 contiguous nucleotides of a DNA having a sequenceselected from the group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQID NOs:1-725, SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ IDNOs:808-830, and SEQ ID NOs:1087-1094, or an RNA transcribed from theDNA, and wherein the double-stranded RNA molecule is at least 50base-pairs in length or is between about 100 to about 500 base-pairs inlength. In an embodiment this invention provides an insecticidalcomposition for controlling a Leptinotarsa species comprising aninsecticidally effective amount of a double-stranded RNA, wherein atleast one strand of the double-stranded RNA is complementary to at least21 contiguous nucleotides of a gene that encodes a ribosomal protein oran RNA transcribed from the gene, wherein the Leptinotarsa species isLeptinotarsa decemlineata, and wherein RNA interference is induced andLeptinotarsa decemlineata mortality occurs, and wherein the ribosomalprotein is a ribosomal L7 protein or a protein encoded by SEQ ID NO:730or wherein the double-stranded RNA comprises a sequence selected fromthe group consisting of SEQ ID NO:989, 988, 1104, or 1105. Relatedaspects of the invention include isolated RNAs of use in the compositionand plants having improved Leptinotarsa resistance provided by treatmentwith the composition.

In various embodiments, the insecticidal composition for controlling aLeptinotarsa species is in the form of at least one selected from thegroup consisting of a solid, liquid (including homogeneous mixtures suchas solutions and non-homogeneous mixtures such as suspensions, colloids,micelles, and emulsions), powder, suspension, emulsion, spray,encapsulated or micro-encapsulation formulation, in or on microbeads orother carrier particulates, in a film or coating, or on or within amatrix, or as a seed treatment. Suitable binders, inert carriers,surfactants, and the like can optionally be included in thepolynucleotide-containing composition, as is known to one skilled informulation of insecticides and seed treatments. The Leptinotarsaspecies to be controlled is generally a species that infests a plant. Insome embodiments, the insecticidal composition is at least oneimplantable formulation selected from the group consisting of aparticulate, pellet, or capsule implanted in the plant; in suchembodiments the method comprises implanting in the plant the implantableformulation. In some embodiments, the insecticidal composition is atleast one in-furrow formulation selected from the group consisting of apowder, granule, pellet, capsule, spray, or drench, or any other formssuited for applying to a furrow; in such embodiments, the methodcomprises an in-furrow treatment with the in-furrow formulation. In oneembodiment the insecticidal composition can be ingested or otherwiseabsorbed internally by the Leptinotarsa species. For example, theinsecticidal composition can be in the form of bait. In someembodiments, the insecticidal composition further comprises one or morecomponents selected from the group consisting of a carrier agent, asurfactant, a cationic lipid (such as that disclosed in Example 18 ofU.S. patent application publication 2011/0296556, incorporated byreference herein), an organosilicone, an organosilicone surfactant, apolynucleotide herbicidal molecule, a non-polynucleotide herbicidalmolecule, a non-polynucleotide pesticide, a safener, and an insectgrowth regulator. In one embodiment the insecticidal composition furthercomprises a nonionic organosilicone surfactant such as SILWET® brandsurfactants, e.g., SILWET L-77® brand surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, currentlyavailable from Momentive Performance Materials, Albany, N.Y. In someembodiments, the insecticidal composition further comprises at least onepesticidal agent selected from the group consisting of a patatin, aplant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidalprotein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidalprotein, a Bacillus laterosporous insecticidal protein, and a Bacillussphaericus insecticidal protein. Alternatively such additionalcomponents or pesticidal agents can be provided separately, e.g., byseparate topical application or by transgenic expression in the plant.Alternatively the plant is topically treated with the insecticidalcomposition as well as with a separate (preceding, following, orconcurrent) application of a substance that improves the efficacy of theinsecticidal composition. For example, a plant can be sprayed with afirst topical application of a solution containing a nonionicorganosilicone surfactant such as SILWET® brand surfactants, e.g.,SILWET L-77® brand surfactant, followed by a second topical applicationof the insecticidal composition, or vice-versa.

It is anticipated that the combination of certain RNAs of use in thismethod (e.g., the dsRNA triggers described in the working Examples) withone or more non-polynucleotide pesticidal agents will result in asynergetic improvement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with the RNA alone orthe non-polynucleotide pesticidal agent alone. In an embodiment, theinsecticidal composition contains one or more RNAs and one or morenon-polynucleotide pesticidal agent selected from the group consistingof a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein,and is found to effect synergistically improved prevention or control ofLeptinotarsa species infestations.

The Leptinotarsa species to be controlled is generally a species thatinfests a plant. The plant can be any plant that is subject toinfestation by a Leptinotarsa species. Of particular interest areembodiments wherein the plant is a solanaceous plant (familySolanaceae). Examples include a plant selected from the group consistingof potato, tomato, and eggplant. Embodiments include those wherein theplant is an ungerminated solanaceous plant seed, a solanaceous plant ina vegetative stage, or a solanaceous plant in a reproductive stage.Embodiments include those wherein the plant is a “seed potato”, meaning,a potato tuber or piece of potato tuber which can be propagated into newpotato plants. In some embodiments, use of the insecticidal compositionresults in control of the Leptinotarsa species, e.g., in growthstunting, increased mortality, decrease in reproductive capacity,decrease in or cessation of feeding behavior or movement, or decrease inor cessation of metamorphosis stage development. In some embodiments,control of the Leptinotarsa species is observed as improved growth orimproved yields of solanaceous plants treated with the insecticidalcomposition, in comparison to plants not treated with the insecticidalcomposition. In some embodiments, control of the Leptinotarsa species isobserved as decreased numbers of eggs, larvae, or adults of theLeptinotarsa species, decreased defoliation or other damage to theplant, or increased yield of harvestable fruit (e.g., tomatoes oreggplants) or tubers (e.g., potatoes).

In various embodiments, the insecticidal composition comprises amicrobial cell or is produced in a microorganism. For example, theinsecticidal composition can include or can be produced in bacteria oryeast cells. In similar embodiments the insecticidal compositioncomprises a transgenic plant cell or is produced in a plant cell (forexample a plant cell transiently expressing the polynucleotide); suchplant cells can be cells in an plant or cells grown in tissue culture orin cell suspension.

The insecticidal composition can be provided for dietary uptake by theLeptinotarsa species by applying the composition to a plant or surfacesubject to infestation by the Leptinotarsa species, for example byspraying, dusting, or coating the plant or a seed of the plant or a seedpotato, or by application of a soil drench or in-furrow treatment, or byproviding in an artificial diet. The insecticidal composition can beprovided for dietary uptake by the Leptinotarsa species in an artificialdiet formulated to meet the particular nutritional requirements formaintaining the Leptinotarsa species, wherein the artificial diet issupplemented with some amount of the RNA obtained from a separate sourcesuch as chemical synthesis or purified from a microbial fermentation;this embodiment can be useful, e.g., for determining the timing andamounts of effective RNA treatment regimes. In some embodiments theinsecticidal composition is provided for dietary uptake by theLeptinotarsa species in the form of a plant cell or in plant cellcomponents, or in a microorganism (such as a bacterium or a yeast) or amicrobial fermentation product, or in a synthetic diet. In oneembodiment the insecticidal composition is provided in the form of baitthat is ingested by the Leptinotarsa species. The insecticidalcomposition can be provided for dietary uptake by the Leptinotarsaspecies in the form of a seed (or seed potato) treatment.

In one embodiment the insecticidal composition is provided in the formof any plant that is subject to infestation by a Leptinotarsa species,wherein the RNA is contained in or on the plant. Such plants can bestably transgenic plants that express the RNA, or non-transgenic plantsthat transiently express the RNA or that have been treated with the RNA,e.g., by spraying or coating. Stably transgenic plants generally containintegrated into their genome a recombinant construct that encodes theRNA. Of particular interest are embodiments wherein the plant is asolanaceous plant (family Solanaceae). Examples include a plant selectedfrom the group consisting of potato, tomato, and eggplant. Embodimentsinclude those wherein the plant is an ungerminated solanaceous plantseed, a solanaceous plant in a vegetative stage, or a solanaceous plantin a reproductive stage. Embodiments include those wherein the plant isa “seed potato”, meaning, a potato tuber or piece of potato tuber whichcan be propagated into new potato plants.

The RNA useful in the insecticidal composition can be single-stranded(ss) or double-stranded (ds). Embodiments include those wherein the RNAis at least one selected from the group consisting of sensesingle-stranded RNA (ssRNA), anti-sense single-stranded (ssRNA), ordouble-stranded RNA (dsRNA); a mixture of RNAs of any of these types canbe used. In one embodiment a double-stranded DNA/RNA hybrid is used. TheRNA can include components other than standard ribonucleotides, e.g., anembodiment is an RNA that comprises terminal deoxyribonucleotides.

The RNA in the insecticidal composition has at least one segment of 18or more contiguous nucleotides with a sequence of about 95% to about100% identity with a fragment of equivalent length of a target gene orDNA having a sequence selected from the Target Gene Sequences Group, orthe DNA complement thereof. In an embodiment the RNA comprises at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of a DNAhaving a sequence selected from the group consisting of the Target GeneSequences Group. In some embodiments, the contiguous nucleotides have asequence of about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identity with a fragment of a DNA having a sequence selectedfrom the Target Gene Sequences Group, or the DNA complement thereof. Insome embodiments the contiguous nucleotides are exactly (100%) identicalto a fragment of equivalent length of a DNA having a sequence selectedfrom the Target Gene Sequences Group, or the DNA complement thereof. Insome embodiments, the RNA has an overall sequence of about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identity with afragment of a DNA having a sequence selected from the Target GeneSequences Group, or the DNA complement thereof.

The RNA in the insecticidal composition comprises at least one segmentof 18 or more contiguous nucleotides with a sequence of about 95% toabout 100% identity with a fragment of equivalent length of a DNA havinga sequence selected from the Target Gene Sequences Group or the DNAcomplement thereof. In some embodiments the RNA comprises at least onesegment of 18 or more contiguous nucleotides, e.g., between 18-24, orbetween 18-28, or between 20-30, or between 20-50, or between 20-100, orbetween 50-100, or between 50-500, or between 100-250, or between100-500, or between 200-1000, or between 500-2000, or even greater. Insome embodiments the segment comprises more than 18 contiguousnucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, orgreater than 30, e.g., about 35, about 40, about 45, about 50, about 55,about 60, about 65, about 70, about 75, about 80, about 85, about 90,about 95, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170, about 180, about 190, about 200, about 210,about 220, about 230, about 240, about 250, about 260, about 270, about280, about 290, about 300, about 350, about 400, about 450, about 500,or greater than 500 contiguous nucleotides. In particular embodiments,the RNA comprises at least one segment of at least 21 contiguousnucleotides with a sequence of 100% identity with a fragment ofequivalent length of a DNA or target gene having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof. Inparticular embodiments, the RNA is a double-stranded nucleic acid (e.g.,dsRNA) with one strand comprising at least one segment of at least 21contiguous nucleotides with a sequence of 100% identity with a fragmentof equivalent length of a DNA or target gene having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof;expressed as base-pairs, such a double-stranded nucleic acid comprisesat least one segment of at least 21 contiguous, perfectly matchedbase-pairs which correspond to a fragment of equivalent length of a DNAor target gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof. In particular embodiments, eachsegment contained in the RNA is of a length greater than that which istypical of naturally occurring regulatory small RNAs, e.g., each segmentis at least about 30 contiguous nucleotides (or base-pairs) in length.In some embodiments, the total length of the RNA, or the length of eachsegment contained in the RNA, is less than the total length of thesequence of interest (DNA or target gene having a sequence selected fromthe group consisting of the Target Gene Sequences Group). In someembodiments, the total length of the RNA is between about 50 to about500 nucleotides (for single-stranded RNAs) or base-pairs (fordouble-stranded RNAs). In some embodiments, the RNA comprises at leastone RNA strand of between about 50 to about 500 nucleotides in length.Embodiments include those in which the RNA comprises at least onesegment having a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the RNA is encoded by a sequence selected from the groupconsisting of SEQ ID NOs:1105-1109.

The RNA in the insecticidal composition is generally designed tosuppress one or more genes (“target genes”). Such target genes caninclude coding or non-coding sequence or both. In specific embodiments,the RNA is designed to suppress one or more target genes, where eachtarget gene has a DNA sequence selected from the group consisting of theTarget Gene Sequences Group. In various embodiments, the RNA is designedto suppress one or more genes, where each gene has a sequence selectedfrom the group consisting of the Target Gene Sequences Group, and can bedesigned to suppress multiple genes from this group, or to targetdifferent regions of one or more of these genes. In an embodiment, theRNA comprises multiple sections or segments each of which comprises atleast one segment of 21 contiguous nucleotides with a sequence of 100%identity with a fragment of equivalent length of a DNA having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof. In such cases, each section can be identical or different insize or in sequence, and can be sense or anti-sense relative to thetarget gene. For example, in one embodiment the RNA can include multiplesections in tandem or repetitive arrangements, wherein each sectioncomprises at least one segment of 21 contiguous nucleotides with asequence of 100% identity with a fragment of equivalent length of a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof; the segments can be from different regions ofthe target gene, e.g., the segments can correspond to different exonregions of the target gene, and “spacer” nucleotides which do notcorrespond to a target gene can optionally be used in between oradjacent to the segments.

The total length of the RNA in the insecticidal composition can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with a fragment of equivalent length of a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof. In other words, the total length of the RNA canbe greater than the length of the section or segment of the RNA designedto suppress one or more target genes, where each target gene has a DNAsequence selected from the group consisting of the Target Gene SequencesGroup. For example, the RNA can have nucleotides flanking the “active”segment of at least one segment of 18 or more contiguous nucleotidesthat suppresses the target gene, or include “spacer” nucleotides betweenactive segments, or can have additional nucleotides at the 5′ end, or atthe 3′ end, or at both the 5′ and 3′ ends. In an embodiment, the RNAcomprises additional nucleotides that are not specifically related(having a sequence not complementary or identical to) to the DNA ortarget gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof, e.g., nucleotides that providestabilizing secondary structure or for convenience in cloning ormanufacturing. In an embodiment, the RNA comprises additionalnucleotides located immediately adjacent to one or more segment of 18 ormore contiguous nucleotides with a sequence of about 95% to about 100%identity with or complementarity to a fragment of equivalent length of aDNA or target gene having a sequence selected from the group consistingof the Target Gene Sequences Group. In an embodiment, the RNA comprisesone such segment, with an additional 5′ G or an additional 3′ C or both,adjacent to the segment. In another embodiment, the RNA is adouble-stranded RNA comprising additional nucleotides to form anoverhang, for example, a dsRNA comprising 2 deoxyribonucleotides to forma 3′ overhang. Thus in various embodiments, the nucleotide sequence ofthe entire RNA is not 100% identical or complementary to a fragment ofcontiguous nucleotides in the DNA or target gene having a sequenceselected from the group consisting of the Target Gene Sequences Group.For example, in some embodiments the RNA comprises at least two segmentseach of 21 contiguous nucleotides with a sequence of 100% identity witha fragment of a DNA having a sequence selected from the Target GeneSequences Group, or the DNA complement thereof, wherein (1) the at leasttwo segments are separated by one or more spacer nucleotides, or (2) theat least two segments are arranged in an order different from that inwhich the corresponding fragments occur in the DNA having a sequenceselected from the Target Gene Sequences Group, or the DNA complementthereof.

In various embodiments the RNA in the insecticidal composition consistsof naturally occurring ribonucleotides. Embodiments include, forexample, synthetic RNAs consisting wholly of ribonucleotides or mainlyof ribonucleotides but with one or more terminal deoxyribonucleotides orone or more terminal dideoxyribonucleotides. In certain embodiments, theRNA comprises non-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In certain embodiments, the RNA comprises chemicallymodified nucleotides. (a) The RNA in the insecticidal composition isprovided by suitable means known to one in the art. Embodiments includethose wherein the RNA is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation.

In some embodiments the RNA is provided as an isolated RNA that is notpart of an expression construct. In some embodiments the RNA is providedas an isolated RNA that is lacking additional elements such as apromoter or terminator sequences. Such RNAs can be relatively short,such as single- or double-stranded RNAs of between about 18 to about 300or between about 50 to about 500 nucleotides (for single-stranded RNAs)or between about 18 to about 300 or between about 50 to about 500base-pairs (for double-stranded RNAs). Alternatively the RNA can beprovided in more complex constructs, e.g., as part of a recombinantexpression construct, or included in a recombinant vector, for examplein a recombinant plant virus vector or in a recombinant baculovirusvector. In some embodiments such recombinant expression constructs orvectors are designed to include additional elements, such as includingadditional RNA encoding an aptamer or ribozyme or an expression cassettefor expressing a gene of interest (e.g., an insecticidal protein).

Methods of Providing Plants Having Improved Resistance to LeptinotarsaSpecies Infestations, and the Plants and Seeds Thus Provided

Another aspect of this invention is directed to a method of providing aplant having improved resistance to a Leptinotarsa species infestationcomprising expressing in the plant at least one polynucleotidecomprising at least one segment of 18 or more contiguous nucleotidesthat is essentially identical or complementary to a fragment of a targetgene or DNA having a sequence selected from the group consisting of theTarget Gene Sequences Group, whereby the resulting plant has improvedresistance to a Leptinotarsa species when compared to a control plant inwhich the polynucleotide is not expressed. In an embodiment, the methodcomprises expressing in the plant at least one polynucleotide comprisingat least one segment of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with a fragment ofequivalent length of a target gene or DNA having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof. Inan embodiment, the invention provides a method of providing a planthaving improved resistance to a Leptinotarsa species infestationcomprising expressing in the plant at least one polynucleotidecomprising at least one segment that is identical or complementary to atleast 21 contiguous nucleotides of a DNA having a sequence selected fromthe group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725,SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094. By “expressing a polynucleotide in the plant” isgenerally meant “expressing an RNA transcript in the plant”, e.g.,expressing in the plant an RNA comprising a ribonucleotide sequence thatis anti-sense or essentially complementary to at least a fragment of atarget gene or DNA having a sequence selected from the group consistingof the Target Gene Sequences Group. Embodiments include those in whichthe polynucleotide expressed in the plant is an RNA comprising at leastone segment having a sequence selected from the group consisting of: SEQID NOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein polynucleotide expressed in the plant is an RNA hairpin encodedby a sequence selected from the group consisting of SEQ IDNOs:1105-1109. Embodiments include those in which the polynucleotideexpressed in the plant comprises a dsRNA with a strand having a sequenceselected from the group consisting of the Trigger Sequences Group.However, the polynucleotide expressed in the plant can also be DNA(e.g., a DNA produced in the plant during genome replication), or theRNA encoded by such DNA. Related aspects of the invention includeisolated polynucleotides of use in the method and plants having improvedLeptinotarsa resistance provided by the method.

The method comprises expressing at least one polynucleotide in a plant,wherein the polynucleotide comprises at least one segment of 18 or morecontiguous nucleotides that is essentially identical or complementary toa fragment of a target gene or DNA having a sequence selected from thegroup consisting of the Target Gene Sequences Group. In someembodiments, a first polynucleotide is provided to a plant in the formof DNA (e.g., in the form of an isolated DNA molecule, or as anexpression construct, or as a transformation vector), and thepolynucleotide expressed in the plant is a second polynucleotide (e.g.,the RNA transcript of the first polynucleotide) in the plant. In anembodiment, the polynucleotide is expressed in the plant by transgenicexpression, i.e., by stably integrating the polynucleotide into theplant's genome from where it can be expressed in a cell or cells of theplant. In an embodiment, a first polynucleotide (e.g., a recombinant DNAconstruct comprising a promoter operably linked to DNA comprising atleast one segment of 18 or more contiguous nucleotides that isessentially identical or complementary to a fragment of a target gene orDNA having a sequence selected from the group consisting of the TargetGene Sequences Group) is stably integrated into the plant's genome fromwhere secondarily produced polynucleotides (e.g., an RNA transcriptcomprising the transcript of the segment of 18 or more contiguousnucleotides that is essentially identical or complementary to a fragmentof a target gene or DNA having a sequence selected from the groupconsisting of the Target Gene Sequences Group) are expressed in a cellor cells of the plant. Methods of providing stably transformed plantsare provided in the section headed “Making and Using Transgenic PlantCells and Transgenic Plants”.

In another embodiment the polynucleotide expressed in the plant isexpressed by transient expression (i.e., expression not resulting fromstable integration of a sequence into the plant's genome). In suchembodiments the method can include a step of introducing apolynucleotide (e.g., dsRNA or dsDNA) into the plant by routinetechniques known in the art. For example, transient expression can beaccomplished by infiltration of a polynucleotide solution using aneedle-less syringe into a leaf of a plant.

In some embodiments where the polynucleotide expressed in the plant isexpressed by transient expression, a first polynucleotide is provided toa plant in the form of RNA or DNA or both RNA and DNA, and a secondarilyproduced second polynucleotide is transiently expressed in the plant. Insome embodiments, the first polynucleotide is one or more selected from:(a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNAmolecule that self-hybridizes to form a double-stranded RNA molecule,(c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNAmolecule (ssDNA), (e) a single-stranded DNA molecule thatself-hybridizes to form a double-stranded DNA molecule, (f) asingle-stranded DNA molecule comprising a modified Pol III gene that istranscribed to an RNA molecule, (g) a double-stranded DNA molecule(dsDNA), (h) a double-stranded DNA molecule comprising a modified PolIII gene that is transcribed to an RNA molecule, and (i) adouble-stranded, hybridized RNA/DNA molecule, or combinations thereof.In specific embodiments, a first polynucleotide is introduced into theplant by topical application to the plant of a polynucleotide-containingcomposition in a suitable form, e.g., as a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or in the form of a treatment of a solanaceous plantseed or treatment of a seed potato. Suitable binders, inert carriers,surfactants, and the like can optionally be included in the composition,as is known to one skilled in formulation of pesticides and seedtreatments. In such embodiments, the polynucleotide-containingcomposition can further include one or more components selected from thegroup consisting of a carrier agent, a surfactant, a cationic lipid(such as that disclosed in Example 18 of U.S. patent applicationpublication 2011/0296556, incorporated by reference herein), anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator;in one embodiment the composition further comprises a nonionicorganosilicone surfactant such as SILWET® brand surfactants, e.g.,SILWET L-77® brand surfactant having CAS Number 27306-78-1 and EPANumber: CAL.REG.NO. 5905-50073-AA, currently available from MomentivePerformance Materials, Albany, N.Y. In some embodiments, the topicallyapplied composition further comprises at least one pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein. Alternatively such additional components orpesticidal agents can be provided separately, e.g., by separate topicalapplication or by transgenic expression in the plant. Alternatively theplant is topically treated with the polynucleotide-containingcomposition as well as with a separate (preceding, following, orconcurrent) application of a substance that improves the efficacy of thepolynucleotide-containing composition. For example, a plant can besprayed with a first topical application of a solution containing anonionic organosilicone surfactant such as SILWET® brand surfactants,e.g., SILWET L-77® brand surfactant, followed by a second topicalapplication of the polynucleotide-containing composition, or vice-versa.

It is anticipated that the combination of certain polynucleotides of usein this method (e.g., the polynucleotide triggers described in theworking Examples) with one or more non-polynucleotide pesticidal agentswill result in a synergetic improvement in prevention or control ofLeptinotarsa species infestations, when compared to the effect obtainedwith the polynucleotide alone or the non-polynucleotide pesticidal agentalone. In an embodiment, a transgenic plant expressing at least onepolynucleotide comprising at least one segment of 18 or more contiguousnucleotides that is essentially identical or complementary to a fragmentof a target gene or DNA having a sequence selected from the groupconsisting of the Target Gene Sequences Group (e.g., the polynucleotidetriggers described in the working Examples) and one or more genesencoding a non-polynucleotide pesticidal agent selected from the groupconsisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein, isfound to exhibit synergistically improved resistance to Leptinotarsaspecies infestations.

In some embodiments where the polynucleotide expressed in the plant isexpressed by transient expression, a first polynucleotide is provided toa plant in the form of RNA or DNA or both RNA and DNA, and a secondarilyproduced second polynucleotide is transiently expressed in the plant;the site of application of the first polynucleotide need not be the samesite where the second polynucleotide is transiently expressed. Forexample, a first polynucleotide can be provided to a plant by topicalapplication onto a leaf, or by injection into a stem, and the secondpolynucleotide can be transiently expressed elsewhere in the plant,e.g., in the roots or throughout the plant. In some embodiments of themethod, a composition comprising at least one polynucleotide istopically applied to above-ground parts of the plant, e.g., sprayed ordusted onto leaves, stems, and flowering parts of the plant. In otherembodiments, a composition comprising at least one polynucleotide istopically applied to below-ground parts of the plant, such as to theroots, e.g., by means of a soil drench. In other embodiments, acomposition comprising at least one polynucleotide is topically appliedto a seed (or, in the case of potatoes, topically applied to a seedpotato) that is grown into the plant having improved resistance to aLeptinotarsa species infestation. In some embodiments the polynucleotideexpressed in the plant is RNA, which can be single-stranded (ss) ordouble-stranded (ds) RNA or a combination of both.

In some embodiments a first polynucleotide (DNA or RNA or both) isprovided to a plant and a second polynucleotide having a sequencecorresponding (identical or complementary) to the first polynucleotideis subsequently expressed in the plant. In such embodiments thepolynucleotide expressed in the plant is an RNA transcript which can bessRNA or dsRNA or a combination of both. In some embodiments where thepolynucleotide is expressed by transient expression, a firstpolynucleotide is provided to a plant in the form of RNA or DNA or bothRNA and DNA, and a secondarily produced second polynucleotide istransiently expressed in the plant; in such embodiments, the firstpolynucleotide one or more selected from: (a) a single-stranded RNAmolecule (ssRNA), (b) a single-stranded RNA molecule thatself-hybridizes to form a double-stranded RNA molecule, (c) adouble-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule(ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to forma double-stranded DNA molecule, (f) a single-stranded DNA moleculecomprising a modified Pol III gene that is transcribed to an RNAmolecule, (g) a double-stranded DNA molecule (dsDNA), (h) adouble-stranded DNA molecule comprising a modified Pol III gene that istranscribed to an RNA molecule, and (i) a double-stranded, hybridizedRNA/DNA molecule, or combinations thereof. In such embodiments where thepolynucleotide is expressed by transient expression the firstpolynucleotide can consist of naturally occurring nucleotides, such asthose which occur in DNA and RNA. In such embodiments where thepolynucleotide is expressed by transient expression the firstpolynucleotide can be chemically modified, or comprises chemicallymodified nucleotides. The first polynucleotide is provided by suitablemeans known to one in the art. Embodiments include those wherein thefirst polynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation. Thefirst polynucleotide can be provided as an RNA or DNA fragment.Alternatively the first polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector; such recombinantexpression constructs or vectors can be designed to include additionalelements, such as expression cassettes for expressing a gene of interest(e.g., an insecticidal protein).

In some embodiments the polynucleotide expressed in the plant is an RNAmolecule and can be relatively short, such as single- or double-strandedRNAs of between about 18 to about 300 or between about 50 to about 500nucleotides (for single-stranded RNAs) or between about 18 to about 300or between about 50 to about 500 base-pairs (for double-stranded RNAs).Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. In some embodimentssuch recombinant expression constructs or vectors are designed toinclude additional elements, such as expression cassettes for expressinga gene of interest (e.g., an insecticidal protein).

The polynucleotide expressed in the plant has at least one segment of 18or more contiguous nucleotides with a sequence of about 95% to about100% identity with a fragment of equivalent length of a DNA having asequence selected from the Target Gene Sequences Group or the DNAcomplement thereof. In an embodiment the polynucleotide expressed in theplant comprises at least one segment of 18 or more contiguousnucleotides that are essentially identical or complementary to afragment of equivalent length of a DNA having a sequence selected fromthe group consisting of the Target Gene Sequences Group. In someembodiments, the contiguous nucleotides have a sequence of about 95%,about 96%, about 97%, about 98%, about 99%, or about 100% identity witha fragment of a DNA having a sequence selected from the Target GeneSequences Group or the DNA complement thereof. In some embodiments thecontiguous nucleotides are exactly (100%) identical to a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In some embodiments,the polynucleotide expressed in the plant has an overall sequence ofabout 95%, about 96%, about 97%, about 98%, about 99%, or about 100%identity with a fragment of a DNA having a sequence selected from theTarget Gene Sequences Group or the DNA complement thereof.

The polynucleotide expressed in the plant is generally designed tosuppress one or more genes (“target genes”). Such target genes caninclude coding or non-coding sequence or both. In specific embodiments,the polynucleotide expressed in the plant is designed to suppress one ormore target genes, where each target gene has a DNA sequence selectedfrom the group consisting of the Target Gene Sequences Group. In variousembodiments, the polynucleotide expressed in the plant is designed tosuppress one or more genes, where each gene has a sequence selected fromthe group consisting of the Target Gene Sequences Group, and can bedesigned to suppress multiple genes from this group, or to targetdifferent regions of one or more of these genes. In an embodiment, thepolynucleotide expressed in the plant comprises multiple sections orsegments each of which comprises at least one segment of 21 contiguousnucleotides with a sequence of 100% identity with a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In such cases, eachsection can be identical or different in size or in sequence, and can besense or anti-sense relative to the target gene. For example, in oneembodiment the polynucleotide expressed in the plant can includemultiple sections in tandem or repetitive arrangements, wherein eachsection comprises at least one segment of 21 contiguous nucleotides witha sequence of 100% identity with a fragment of equivalent length of aDNA having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof the segments can be from different regions ofthe target gene, e.g., the segments can correspond to different exonregions of the target gene, and “spacer” nucleotides which do notcorrespond to a target gene can optionally be used in between oradjacent to the segments.

The total length of the polynucleotide expressed in the plant can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with a fragment of equivalent length of a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof. In other words, the total length of thepolynucleotide expressed in the plant can be greater than the length ofthe section or segment of the polynucleotide designed to suppress one ormore target genes, where each target gene has a DNA sequence selectedfrom the group consisting of the Target Gene Sequences Group. Forexample, the polynucleotide expressed in the plant can have nucleotidesflanking the “active” segment of at least one segment of 18 or morecontiguous nucleotides that suppresses the target gene, or include“spacer” nucleotides between active segments, or can have additionalnucleotides at the 5′ end, or at the 3′ end, or at both the 5′ and 3′ends. In an embodiment, the polynucleotide expressed in the plantcomprises additional nucleotides that are not specifically related(i.e., having a sequence not complementary or identical to) to the DNAor target gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof, e.g., nucleotides that providestabilizing secondary structure or for convenience in cloning ormanufacturing. In an embodiment, the polynucleotide expressed in theplant comprises additional nucleotides located immediately adjacent toone or more segment of 18 or more contiguous nucleotides with a sequenceof about 95% to about 100% identity with or complementarity to afragment of equivalent length of a DNA or target gene having a sequenceselected from the group consisting of the Target Gene Sequences Group.In an embodiment, the polynucleotide expressed in the plant comprisesone such segment, with an additional 5′ G or an additional 3′ C or both,adjacent to the segment. In another embodiment, the polynucleotideexpressed in the plant is a double-stranded RNA comprising additionalnucleotides to form an overhang, for example, a dsRNA comprising 2deoxyribonucleotides to form a 3′ overhang. Thus in various embodiments,the nucleotide sequence of the entire polynucleotide expressed in theplant is not 100% identical or complementary to a fragment of contiguousnucleotides in the DNA or target gene having a sequence selected fromthe group consisting of the Target Gene Sequences Group. For example, insome embodiments the polynucleotide expressed in the plant comprises atleast two segments each of 21 contiguous nucleotides with a sequence of100% identity with a fragment of a DNA having a sequence selected fromthe Target Gene Sequences Group, or the DNA complement thereof, wherein(1) the at least two segments are separated by one or more spacernucleotides, or (2) the at least two segments are arranged in an orderdifferent from that in which the corresponding fragments occur in theDNA having a sequence selected from the Target Gene Sequences Group, orthe DNA complement thereof.

In a related aspect, this invention is directed to the plant havingimproved resistance to a Leptinotarsa species infestation, provided byexpressing in the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of a DNAhaving a sequence selected from the group consisting of the Target GeneSequences Group, whereby the resulting plant has improved resistance toa Leptinotarsa species infestation when compared to a control plant inwhich the polynucleotide is not expressed. In a related aspect, thisinvention is directed to the plant having improved resistance to aLeptinotarsa species infestation, provided by expressing in the plant atleast one polynucleotide comprising at least one segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity with a fragment of equivalent length of a DNA having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof, whereby the resulting plant has improved resistance to aLeptinotarsa species infestation when compared to a control plant inwhich the polynucleotide is not expressed. An embodiment is asolanaceous plant having improved resistance to a Leptinotarsa speciesinfestation when compared to a control plant, provided by expressing inthe plant an RNA having a sequence selected from the group consistingof: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114, or the complementthereof, or expressing in the plant an RNA hairpin encoded by a sequenceselected from the group consisting of SEQ ID NOs:1105-1109. In yetanother aspect, this invention is directed to seed (especiallytransgenic progeny seed) produced by the plant having improvedresistance to a Leptinotarsa species infestation, as provided by thismethod. Also contemplated is a commodity product produced by the planthaving improved resistance to a Leptinotarsa species infestation, asprovided by this method, and a commodity product produced from thetransgenic progeny seed of such a plant.

Recombinant DNA Constructs for Controlling a Leptinotarsa Species

Another aspect of this invention provides a recombinant DNA constructcomprising a heterologous promoter operably linked to a DNA elementcomprising at least one segment of 18 or more contiguous nucleotideswith a sequence of about 95% to about 100% identity with a fragment of aDNA having a sequence selected from the Target Gene Sequences Group, orthe DNA complement thereof. In some embodiments, the recombinant DNAconstruct comprises a heterologous promoter operably linked to: (a) DNAcomprising a nucleotide sequence that is complementary to at least 21contiguous nucleotides of a target gene having a sequence selected fromthe group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725,SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or an RNA transcribed from the target gene; or (b) a DNAcomprising 21 or more contiguous nucleotides having 100% identity to afragment of equivalent length of a DNA having a sequence selected fromthe group consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725,SEQ ID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094, or the DNA complement thereof; or (c) DNA encoding atleast one silencing element that is complementary to at least 21contiguous nucleotides of a target gene or an RNA transcribed from thetarget gene, wherein the target gene has a sequence selected from thegroup consisting of: SEQ ID NO:730, SEQ ID NO:807, SEQ ID NOs:1-725, SEQID NOs:726-729, SEQ ID NOs:731-806, SEQ ID NOs:808-830, and SEQ IDNOs:1087-1094; or (d) DNA encoding at least one silencing elementcomprising at least 21 contiguous nucleotides that are complementary toa target gene selected from the genes in the Target Gene Sequences Groupor an RNA transcribed from the target gene; or (e) DNA encoding a RNAcomprising at least 21 contiguous nucleotides that are complementary toa nucleotide sequence selected from the Trigger Sequences Group, or thecomplement thereof, or an orthologous nucleotide sequence from aLeptinotarsa species or a Tribolium species, wherein the orthologousnucleotide sequence has at least 95% sequence identity with a nucleotidesequence selected from the Trigger Sequences Group, wherein thepercentage sequence identity is calculated over the same length; or (f)DNA encoding a RNA comprising at least one double-stranded RNA region,at least one strand of which comprises at least 21 contiguousnucleotides that are complementary to a nucleotide sequence selectedfrom the Trigger Sequences Group, or the complement thereof, or anorthologous nucleotide sequence from a Leptinotarsa species or aTribolium species, wherein the orthologous nucleotide sequence has atleast 95% sequence identity with a nucleotide sequence selected from thegroup consisting of the Trigger Sequences Group, wherein the percentagesequence identity is calculated over the same length; or (g) DNAencoding RNA comprising a nucleotide sequence selected from the TriggerSequences Group, or the complement thereof. Embodiments include arecombinant DNA construct comprising a heterologous promoter operablylinked to a DNA element encoding an RNA having a sequence selected fromthe group consisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114,or the complement thereof, or comprising a heterologous promoteroperably linked to a DNA element encoding an RNA hairpin encoded by asequence selected from the group consisting of SEQ ID NOs:1105-1109.Embodiments include a recombinant DNA construct comprising aheterologous promoter operably linked to a DNA encoding a dsRNA with astrand having a sequence selected from the group consisting of theTrigger Sequences Group. The recombinant DNA constructs are useful inproviding a plant having improved resistance to a Leptinotarsa speciesinfestation, e.g., by expressing in a plant a transcript of such arecombinant DNA construct. The recombinant DNA constructs are alsouseful in the manufacture of polynucleotides useful in makingcompositions that can be applied to a plant, seed, propagatable plantpart, soil or field, or surface in need of protection from aLeptinotarsa species infestation. Related aspects of the inventioninclude: compositions comprising the recombinant DNA construct; a plantchromosome or a plastid or a recombinant plant virus vector or arecombinant baculovirus vector comprising the recombinant DNA construct;a transgenic solanaceous plant cell having in its genome the recombinantDNA construct, optionally comprising in its genome DNA encoding at leastone pesticidal agent selected from the group consisting of a patatin, aplant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidalprotein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidalprotein, a Bacillus laterosporous insecticidal protein, and a Bacillussphaericus insecticidal protein, and a transgenic solanaceous plantincluding such a transgenic solanaceous plant cell, or a fruit, seed, orpropagatable part of the transgenic solanaceous plant; and plants havingimproved Leptinotarsa resistance provided by expression of or treatmentwith the recombinant DNA construct or the RNA encoded therein.

The recombinant DNA construct comprises a heterologous promoter operablylinked to DNA comprising at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with afragment of equivalent length of a DNA having a sequence selected fromthe Target Gene Sequences Group or the DNA complement thereof. In someembodiments, the segment of 18 or more contiguous nucleotides has asequence with about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identity with a fragment of a DNA having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof. Insome embodiments the contiguous nucleotides are exactly (100%) identicalto a fragment of equivalent length of a DNA having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof. Insome embodiments, the DNA has an overall sequence of about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identity with a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof.

The recombinant DNA construct therefore comprises a heterologouspromoter operably linked to DNA comprising at least one segment of 18 ormore contiguous nucleotides designed to suppress expression of a targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof. In some embodiments the DNA comprises atleast one segment of 18 or more contiguous nucleotides, e.g., between18-24, or between 18-28, or between 20-30, or between 20-50, or between20-100, or between 50-100, or between 50-500, or between 100-250, orbetween 100-500, or between 200-1000, or between 500-2000, or evengreater. In some embodiments the segment comprises more than 18contiguous nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or greater than 30, e.g., about 35, about 40, about 45, about50, about 55, about 60, about 65, about 70, about 75, about 80, about85, about 90, about 95, about 100, about 110, about 120, about 130,about 140, about 150, about 160, about 170, about 180, about 190, about200, about 210, about 220, about 230, about 240, about 250, about 260,about 270, about 280, about 290, about 300, about 350, about 400, about450, about 500, or greater than 500 contiguous nucleotides. Inparticular embodiments, the DNA encodes an RNA containing at least onesegment of at least 21 contiguous nucleotides with a sequence of 100%identity with a fragment of equivalent length of a DNA or target genehaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof. In particular embodiments, the DNA encodes adouble-stranded nucleic acid (e.g., dsRNA) with one strand comprising atleast one segment of at least 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of equivalent length of a DNA or targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof; expressed as base-pairs, such adouble-stranded nucleic acid comprises at least one segment of at least21 contiguous, perfectly matched base-pairs which correspond to afragment of equivalent length of a DNA or target gene having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof. In particular embodiments, each segment contained in the DNA isof a length greater than that which is typical of naturally occurringregulatory small RNAs. In some embodiments, each segment is at leastabout 30 contiguous nucleotides (or base-pairs) in length. In someembodiments, the total length of the DNA, or the length of each segmentcontained in the polynucleotide, is less than the total length of thesequence of interest (DNA or target gene having a sequence selected fromthe group consisting of the Target Gene Sequences Group). In someembodiments, the total length of the DNA is between about 50 to about500. In some embodiments, the DNA encodes an RNA having a sequenceselected from the group consisting of: SEQ ID NOs:831-1085, 1095-1104,and 1110-1114, or the complement thereof. In some embodiments, therecombinant DNA construct comprises a sequence selected from the groupconsisting of SEQ ID NOs:1105-1109.

The recombinant DNA construct comprises a heterologous promoter operablylinked to DNA generally designed to suppress one or more genes (“targetgenes”). Such target genes can include coding or non-coding sequence orboth. In specific embodiments, the recombinant DNA construct is designedto suppress one or more target genes, where each target gene has a DNAsequence selected from the group consisting of the Target Gene SequencesGroup. In various embodiments, the recombinant DNA construct is designedto suppress one or more genes, where each gene has a sequence selectedfrom the group consisting of the Target Gene Sequences Group, and can bedesigned to suppress multiple genes from this group, or to targetdifferent regions of one or more of these genes. In an embodiment, therecombinant DNA construct comprises a heterologous promoter operablylinked to multiple sections or segments each of which comprises at leastone segment of 21 contiguous nucleotides with a sequence of 100%identity with a fragment of equivalent length of a DNA having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof. In such cases, each section can be identical or different insize or in sequence, and can be sense or anti-sense relative to thetarget gene. For example, in one embodiment the recombinant DNAconstruct can include a heterologous promoter operably linked tomultiple sections in tandem or repetitive arrangements, wherein eachsection comprises at least one segment of 21 contiguous nucleotides witha sequence of 100% identity with a fragment of equivalent length of aDNA having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof the segments can be from different regions ofthe target gene, e.g., the segments can correspond to different exonregions of the target gene, and “spacer” nucleotides which do notcorrespond to a target gene can optionally be used in between oradjacent to the segments.

The recombinant DNA construct comprises a heterologous promoter operablylinked to DNA which can have a total length that is greater than 18contiguous nucleotides, and can include nucleotides in addition to thesegment of at least one segment of 18 or more contiguous nucleotideshaving the sequence of about 95% to about 100% identity with a fragmentof equivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In other words, thetotal length of the DNA can be greater than the length of the segment ofthe DNA designed to suppress one or more target genes, where each targetgene has a DNA sequence selected from the group consisting of the TargetGene Sequences Group. For example, the DNA can have nucleotides flankingthe “active” segment of at least one segment of 18 or more contiguousnucleotides that suppresses the target gene, or include “spacer”nucleotides between active segments, or can have additional nucleotidesat the 5′ end, or at the 3′ end, or at both the 5′ and 3′ ends. In anembodiment, the heterologous promoter is operably linked to DNAcomprising additional nucleotides that are not specifically related(having a sequence not complementary or identical to) to the DNA ortarget gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof, e.g., nucleotides that providestabilizing secondary structure or for convenience in cloning ormanufacturing. In an embodiment, the heterologous promoter is operablylinked to DNA comprising additional nucleotides located immediatelyadjacent to one or more segment of 18 or more contiguous nucleotideswith a sequence of about 95% to about 100% identity with orcomplementarity to a fragment of equivalent length of a DNA or targetgene having a sequence selected from the group consisting of the TargetGene Sequences Group. In an embodiment, the heterologous promoter isoperably linked to DNA comprising one such segment, with an additional5′ G or an additional 3′ C or both, adjacent to the segment. In anotherembodiment, the heterologous promoter is operably linked to DNA encodinga double-stranded RNA comprising additional nucleotides to form anoverhang. Thus in various embodiments, the nucleotide sequence of theentire DNA operably linked to the heterologous promoter is not 100%identical or complementary to a fragment of contiguous nucleotides inthe DNA or target gene having a sequence selected from the groupconsisting of the Target Gene Sequences Group. For example, in someembodiments the heterologous promoter is operably linked to DNAcomprising at least two segments each of 21 contiguous nucleotides witha sequence of 100% identity with a fragment of a DNA having a sequenceselected from the Target Gene Sequences Group, or the DNA complementthereof, wherein (1) the at least two segments are separated by one ormore spacer nucleotides, or (2) the at least two segments are arrangedin an order different from that in which the corresponding fragmentsoccur in the DNA having a sequence selected from the Target GeneSequences Group, or the DNA complement thereof.

In recombinant DNA constructs, the heterologous promoter is operablylinked to DNA that encodes a transcript that can be single-stranded (ss)or double-stranded (ds) or a combination of both. Embodiments of themethod include those wherein the DNA encodes a transcript comprisingsense single-stranded RNA (ssRNA), anti-sense ssRNA, or double-strandedRNA (dsRNA), or a combination of any of these.

The recombinant DNA construct is provided by suitable means known to onein the art. Embodiments include those wherein the recombinant DNAconstruct is synthesized in vitro, produced by expression in amicroorganism or in cell culture (such as plant or insect cells grown inculture), produced by expression in a plant cell, or produced bymicrobial fermentation.

The heterologous promoter of use in recombinant DNA constructs isselected from the group consisting of a promoter functional in a plant,a promoter functional in a prokaryote, a promoter functional in a fungalcell, and a baculovirus promoter. Non-limiting examples of promoters aredescribed in the section headed “Promoters”.

In some embodiments, the recombinant DNA construct comprises a secondpromoter also operably linked to the DNA. For example, the DNAcomprising at least one segment of 18 or more contiguous nucleotides canbe flanked by two promoters arranged so that the promoters transcribe inopposite directions and in a convergent manner, yielding opposite-strandtranscripts of the DNA that are complementary to and capable ofhybridizing with each other to form double-stranded RNA. In oneembodiment, the DNA is located between two root-specific promoters,which enable transcription of the DNA in opposite directions, resultingin the formation of dsRNA.

In some embodiments the recombinant DNA construct comprises other DNAelements in addition to the heterologous promoter operably linked to DNAcomprising at least one segment of 18 or more contiguous nucleotideswith a sequence of about 95% to about 100% identity with a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. Such DNA elementsare known in the art, and include but are not limited to introns,recombinase recognition sites, aptamers or ribozymes, additional andadditional expression cassettes for expressing coding sequences (e.g.,to express a transgene such as an insecticidal protein or selectablemarker) or non-coding sequences (e.g., to express additional suppressionelements). Inclusion of one or more recognition sites for binding andcleavage by a small RNA (e.g., by a miRNA or an siRNA that is expressedonly in a particular cell or tissue) allows for more precise expressionpatterns in a plant, wherein the expression of the recombinant DNAconstruct is suppressed where the small RNA is expressed.

In some embodiments, the recombinant DNA construct is provided in arecombinant vector. By “recombinant vector” is meant a recombinantpolynucleotide molecule that is used to transfer genetic informationfrom one cell to another. Embodiments suitable to this inventioninclude, but are not limited to, recombinant plasmids, recombinantcosmids, artificial chromosomes, and recombinant viral vectors such asrecombinant plant virus vectors and recombinant baculovirus vectors.Alternative embodiments include recombinant plasmids, recombinantcosmids, artificial chromosomes, and recombinant viral vectors such asrecombinant plant virus vectors and recombinant baculovirus vectorscomprising the DNA element without the heterologous promoter.

In some embodiments, the recombinant DNA construct is provided in aplant chromosome or plastid, e.g., in a transgenic plant cell or atransgenic plant. Thus, also encompassed by this invention is atransgenic plant cell having in its genome the recombinant DNAconstruct, as well as a transgenic plant or partially transgenic plantincluding such a transgenic plant cell. Partially transgenic plantsinclude, e.g., a non-transgenic scion grafted onto a transgenicrootstock including the transgenic plant cell. Embodiments include atransgenic tomato rootstock including the transgenic plant cell. Theplant can be any plant that is subject to infestation by a Leptinotarsaspecies. Of particular interest are embodiments wherein the plant is asolanaceous plant (family Solanaceae). Examples include a plant selectedfrom the group consisting of potato, tomato, and eggplant. Embodimentsinclude those wherein the plant is an ungerminated solanaceous plantseed, a solanaceous plant in a vegetative stage, or a solanaceous plantin a reproductive stage. Embodiments include those wherein the plant isa “seed potato”, meaning a potato tuber or piece of potato tuber whichcan be propagated into new potato plants. In yet another aspect, thisinvention is directed to seed (especially transgenic progeny seed)produced by the transgenic plant having in its genome a recombinant DNAconstruct as described herein. Embodiments also encompass a transgenicseed potato having in its genome a recombinant DNA construct asdescribed herein. Also contemplated is a commodity product produced bysuch a transgenic plant, and a commodity product produced from thetransgenic progeny seed of such a transgenic plant.

The recombinant DNA construct can be provided in a composition fortopical application to a surface of a plant or of a plant seed, or fortopical application to any substrate needing protection from aLeptinotarsa species infestation. Likewise, the recombinant DNAconstruct can be provided in a composition for topical application to aLeptinotarsa species, or in a composition for ingestion by aLeptinotarsa species. In various embodiments, such compositionscontaining the recombinant DNA construct are provided in the form of atleast one selected from the group consisting of a solid, liquid(including homogeneous mixtures such as solutions and non-homogeneousmixtures such as suspensions, colloids, micelles, and emulsions),powder, suspension, emulsion, spray, encapsulated or micro-encapsulationformulation, in or on microbeads or other carrier particulates, in afilm or coating, or on or within a matrix, or as a seed treatment. Thetopical application can be in the form of topical treatment of fruits ofsolanaceous plants or seeds from fruits of solanaceous plants, or in theform of topical treatment of “seed potato” tubers or pieces of tuber(e.g., by soaking, coating, or dusting the seed potato). Suitablebinders, inert carriers, surfactants, and the like can be included inthe composition containing the recombinant DNA construct, as is known toone skilled in formulation of pesticides and seed treatments. In someembodiments, the composition for topical application containing therecombinant DNA construct is at least one topically implantableformulation selected from the group consisting of a particulate, pellet,or capsule topically implanted in the plant; in such embodiments themethod comprises topically implanting in the plant the topicallyimplantable formulation. In some embodiments, the composition fortopical application containing the recombinant DNA construct is at leastone in-furrow formulation selected from the group consisting of apowder, granule, pellet, capsule, spray, or drench, or any other formssuited for topically applying to a furrow; in such embodiments, themethod includes an in-furrow treatment with the in-furrow formulation.In one embodiment the composition for topical application containing therecombinant DNA construct can be ingested or otherwise absorbedinternally by the Leptinotarsa species. For example, the composition fortopical application containing the recombinant DNA construct can be inthe form of bait. In some embodiments, the composition containing therecombinant DNA construct further comprises one or more componentsselected from the group consisting of a carrier agent, a surfactant, acationic lipid (such as that disclosed in Example 18 of U.S. patentapplication publication 2011/0296556, incorporated by reference herein),an organosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator.In one embodiment the composition containing the recombinant DNAconstruct further comprises a nonionic organosilicone surfactant such asSILWET® brand surfactants, e.g., SILWET L-77® brand surfactant havingCAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA,currently available from Momentive Performance Materials, Albany, N.Y.In some embodiments, the composition containing the recombinant DNAconstruct further comprises at least one pesticidal agent selected fromthe group consisting of a patatin, a plant lectin, a phytoecdysteroid, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein.

It is anticipated that the combination of certain recombinant DNAconstructs as described herein (e.g., recombinant DNA constructsincluding the polynucleotide triggers described in the workingExamples), whether transgenically expressed or topically applied, withone or more non-polynucleotide pesticidal agents, whether transgenicallyexpressed or topically applied, will result in a synergetic improvementin prevention or control of Leptinotarsa species infestations, whencompared to the effect obtained with the recombinant DNA constructsalone or the non-polynucleotide pesticidal agent alone. In anembodiment, a recombinant DNA construct for expressing one or morepolynucleotides as well as one or more genes encoding anon-polynucleotide pesticidal agent selected from the group consistingof a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein, isfound to provide synergistically improved resistance to Leptinotarsaspecies infestations in plants expressing the recombinant DNA construct.An embodiment relates to a recombinant DNA construct for expressing anRNA comprising a segment having a sequence selected from the groupconsisting of SEQ ID NOs:831-1085 and 1095 as well as one or more genesencoding a non-polynucleotide pesticidal agent selected from the groupconsisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein.

The composition containing the recombinant DNA construct can be providedfor dietary uptake by a Leptinotarsa species by applying the compositionto a plant or surface subject to infestation by the Leptinotarsaspecies, for example by spraying, dusting, or coating the plant, or byapplication of a soil drench, or by providing in an artificial diet. Thecomposition containing the recombinant DNA construct can be provided fordietary uptake by a Leptinotarsa species in an artificial dietformulated to meet the particular nutritional requirements formaintaining the Leptinotarsa species, wherein the artificial diet issupplemented with some amount of the recombinant DNA construct obtainedfrom a separate source such as in vitro synthesis or purified from amicrobial fermentation or other biological source; this embodiment canbe useful, e.g., for determining the timing and amounts of effectivetreatment regimes. In some embodiments the composition containing therecombinant DNA construct is provided for dietary uptake by theLeptinotarsa species in the form of a plant cell or in plant cellcomponents, or in a microorganism (such as a bacterium or a yeast) or amicrobial fermentation product, or in a synthetic diet. In oneembodiment the composition containing the recombinant DNA construct isprovided in the form of bait that is ingested by the Leptinotarsaspecies. The composition containing the recombinant DNA construct can beprovided for dietary uptake by the Leptinotarsa species in the form of aseed treatment.

In various embodiments, the composition containing the recombinant DNAconstruct comprises a microbial cell or is produced in a microorganism.For example, the composition for containing the recombinant DNAconstruct can include or can be produced in bacteria or yeast cells. Insimilar embodiments the composition containing the recombinant DNAconstruct comprises a transgenic plant cell or is produced in a plantcell (for example a plant cell transiently expressing the recombinantDNA construct); such plant cells can be cells in an plant or cells grownin tissue culture or in cell suspension.

Transgenic Solanaceous Plant Cells

Several embodiments relate to transgenic solanaceous plant cellsexpressing a polynucleotide useful in the methods described herein forsuppressing expression of a target gene in a Leptinotarsa species or forcontrolling a Leptinotarsa infestation. In one aspect this inventionprovides a transgenic solanaceous plant cell having in its genome arecombinant DNA encoding RNA comprising at least one segment of 18 ormore contiguous nucleotides with a sequence of about 95% to about 100%identity with a fragment of a DNA having a sequence selected from theTarget Gene Sequences Group, or the DNA complement thereof. In oneaspect this invention provides a transgenic solanaceous plant cellhaving in its genome a recombinant DNA encoding RNA comprising at leastone silencing element essentially identical or essentially complementaryto a fragment of a target gene sequence of the Leptinotarsa specieslarvae, wherein the target gene sequence is selected from the TargetGene Sequences Group, or the DNA complement thereof. In one aspect thisinvention provides a transgenic solanaceous plant cell having in itsgenome a recombinant DNA encoding RNA that suppresses expression of atarget gene in a Leptinotarsa species that contacts or ingests the RNA,wherein the RNA comprises at least one silencing element having at leastone segment of 18 or more contiguous nucleotides complementary to afragment of the target gene, and wherein the target gene is selectedfrom the group consisting of the genes in the Target Gene SequencesGroup. A specific embodiment is a transgenic solanaceous plant cellhaving in its genome a recombinant DNA encoding RNA that suppressesexpression of a target gene in a Leptinotarsa species that contacts oringests the RNA, wherein the RNA comprises at least one silencingelement having at least one segment of 18 or more contiguous nucleotidescomplementary to a fragment of one or more exocyst target genes;suitable exocyst target genes include the Leptinotarsa exocyst genesprovided in Table 4 or homologous sequences identified from other insectspecies. In one aspect this invention provides a transgenic solanaceousplant cell having in its genome a recombinant DNA encoding an RNA havinga sequence selected from the group consisting of: SEQ ID NOs:831-1085,1095-1104, and 1110-1114, or the complement thereof, or a recombinantDNA selected from the group consisting of SEQ ID NOs:1105-1109;embodiments include a transgenic solanaceous plant cell having in itsgenome a recombinant DNA encoding a dsRNA with a strand having asequence selected from the group consisting of the Trigger SequencesGroup. Such transgenic solanaceous plant cells are useful in providing atransgenic solanaceous plant having improved resistance to aLeptinotarsa species infestation when compared to a control plantlacking such plant cells. The transgenic solanaceous plant cell can anisolated transgenic solanaceous plant cell, or a transgenic solanaceousplant cell grown in culture, or a transgenic cell of any transgenicsolanaceous plant that is subject to infestation by a Leptinotarsaspecies. Examples include a transgenic solanaceous plant selected fromthe group consisting of potato, tomato, and eggplant. Embodimentsinclude those wherein the transgenic solanaceous plant is anungerminated transgenic solanaceous plant seed, a transgenic solanaceousplant in a vegetative stage, or a transgenic solanaceous plant in areproductive stage. Embodiments include those wherein the transgenicsolanaceous plant is a potato tuber or piece of potato tuber (“seedpotato”) which can be propagated into new transgenic potato plants.

In an embodiment, the recombinant DNA is stably integrated into thetransgenic solanaceous plant's genome from where it can be expressed ina cell or cells of the transgenic solanaceous plant. Methods ofproviding stably transformed plants are provided in the section headed“Making and Using Transgenic Plant Cells and Transgenic Plants”.

Several embodiments relate to a transgenic solanaceous plant cell havingin its genome a recombinant DNA encoding RNA that suppresses expressionof a target gene in a Leptinotarsa species that contacts or ingests theRNA, wherein the RNA comprises at least one silencing elementcomplementary to the target gene, and wherein the target gene sequenceis selected from the Target Gene Sequences Group or the complementthereof. In some embodiments, the silencing element comprises at leastone 18 or more contiguous nucleotides with a sequence of about 95% toabout 100% complementarity to a fragment of equivalent length of a DNAhaving a sequence selected from the group consisting of the Target GeneSequences Group. In some embodiments, the silencing element comprises atleast one 18 or more contiguous nucleotides capable of hybridizing invivo or of hybridizing under physiological conditions (e.g., such asphysiological conditions normally found in the cells of a Leptinotarsaspecies) to a fragment of equivalent length of a DNA having a sequenceselected from the group consisting of the Target Gene Sequences Group.The contiguous nucleotides number at least 18, e.g., between 18-24, orbetween 18-28, or between 20-30, or between 20-50, or between 20-100, orbetween 50-100, or between 50-500, or between 100-250, or between100-500, or between 200-1000, or between 500-2000, or even greater. Insome embodiments, the contiguous nucleotides number more than 18, e.g.,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30,e.g., about 35, about 40, about 45, about 50, about 55, about 60, about65, about 70, about 75, about 80, about 85, about 90, about 95, about100, about 110, about 120, about 130, about 140, about 150, about 160,about 170, about 180, about 190, about 200, about 210, about 220, about230, about 240, about 250, about 260, about 270, about 280, about 290,about 300, about 350, about 400, about 450, about 500, or greater than500 contiguous nucleotides. In particular embodiments, the silencingelement comprises at least one segment of at least 21 contiguousnucleotides with a sequence of 100% identity with a fragment ofequivalent length of a DNA or target gene having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof. Inparticular embodiments, the RNA is a double-stranded nucleic acid (e.g.,dsRNA) with one strand comprising at least one segment of at least 21contiguous nucleotides with a sequence of 100% identity with a fragmentof equivalent length of a DNA or target gene having a sequence selectedfrom the Target Gene Sequences Group or the DNA complement thereof;expressed as base-pairs, such a double-stranded nucleic acid comprisesat least one segment of at least 21 contiguous, perfectly matchedbase-pairs which correspond to a fragment of equivalent length of a DNAor target gene having a sequence selected from the Target Gene SequencesGroup or the DNA complement thereof. In particular embodiments, eachsilencing element contained in the RNA is of a length greater than thatwhich is typical of naturally occurring regulatory small RNAs. In someembodiments, each segment is at least about 30 contiguous nucleotides(or base-pairs) in length. In particular embodiments, the RNA is betweenabout 50 to about 500 nucleotides in length. In particular embodiments,the RNA has a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orthe RNA is encoded by a sequence selected from the group consisting ofSEQ ID NOs:1105-1109.

In some embodiments, the transgenic solanaceous plant cell is furthercapable expressing additional heterologous DNA sequences. In anembodiment, the transgenic solanaceous plant cell has a genome thatfurther comprises recombinant DNA encoding at least one pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein. In particular embodiments,the transgenic solanaceous plant cell has stably integrated in itsgenome (i) recombinant DNA encoding at least one RNA with a sequenceselected from the group consisting of SEQ ID NOs:831-1085 and 1095 and(ii) DNA encoding at least one pesticidal agent selected from the groupconsisting of a patatin, a plant lectin, a phytoecdysteroid, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein.

In a related aspect, this invention is directed to a transgenicsolanaceous plant including the transgenic solanaceous plant cell, acommodity product produced from the transgenic solanaceous plant, andtransgenic progeny solanaceous plant seed or transgenic propagatablepart of the transgenic solanaceous plant. Embodiments include atransgenic tomato plant, a transgenic tomato rootstock, a transgeniceggplant, or a transgenic potato plant having improved resistance to aLeptinotarsa species infestation. Also contemplated is a commodityproduct produced by the transgenic solanaceous plant, and a commodityproduct produced from the transgenic progeny seed of such a transgenicsolanaceous plant.

Insecticidal Compositions for Controlling Leptinotarsa Species

Another aspect of this invention provides an insecticidal compositionfor controlling a Leptinotarsa species, wherein the insecticidalcomposition consists essentially of an RNA molecule that causesmortality or stunting of growth in a Leptinotarsa species when ingestedor contacted by the Leptinotarsa species, and wherein the RNA moleculecomprises at least one segment of 18 or more contiguous nucleotides thatis essentially complementary to a fragment of a DNA having a sequenceselected from the Target Gene Sequences Group, or the DNA complementthereof. In this context “controlling” a Leptinotarsa species comprisesinducement of a physiological or behavioural change in a Leptinotarsaspecies (adult or larvae) such as, but not limited to, growth stuntingor increased mortality. In some embodiments, “controlling” aLeptinotarsa species is achieved by a decrease in reproductive capacity,decrease in or cessation of feeding behavior or movement, or decrease inor cessation of metamorphosis stage development in a Leptinotarsaspecies. Generally the RNA molecule has been isolated, that is,substantially purified from a mixture such as from a fermentation orfrom an in vitro synthesis mixture. In one embodiment the RNA moleculecomprises at least one segment of 18 or more contiguous nucleotides witha sequence of about 95% to about 100% complementarity to a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In some embodimentsthe RNA molecule comprises at least one segment of 18 or more contiguousnucleotides that is essentially complementary to a fragment of a DNAhaving a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orwherein the RNA molecule is encoded by a sequence selected from thegroup consisting of SEQ ID NOs:1105-1109. In some embodiments the RNAmolecule is double-stranded, and the at least one segment is betweenabout 50 to about 500 base-pairs in length. In some embodiments the RNAmolecule is a dsRNA with a strand having a sequence selected from thegroup consisting of the Trigger Sequences Group. In some embodiments, aninsecticidal composition is provided for controlling a Leptinotarsaspecies, wherein the insecticidal composition comprises adouble-stranded RNA, wherein at least one strand of the double-strandedRNA is complementary to at least 21 contiguous nucleotides of a genethat encodes a ribosomal protein or an RNA transcribed from the gene,wherein the Leptinotarsa species is Leptinotarsa decemlineata, andwherein RNA interference is induced and Leptinotarsa decemlineatamortality occurs, and wherein the ribosomal protein is a ribosomal L7protein or a protein encoded by SEQ ID NO:730 or wherein thedouble-stranded RNA comprises a sequence selected from the groupconsisting of SEQ ID NO:989, 988, 1104, or 1105.

Embodiments of the RNA molecule include those wherein the segment of 18or more contiguous nucleotides has a sequence with about 95%, about 96%,about 97%, about 98%, about 99%, or about 100% complementarity to afragment of a DNA having a sequence selected from the Target GeneSequences Group or the DNA complement thereof. In some embodiments thecontiguous nucleotides are exactly (100%) complementary to a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In some embodiments,the RNA molecule has an overall sequence of about 95%, about 96%, about97%, about 98%, about 99%, or about 100% complementarity with a DNAhaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof.

Embodiments of the RNA molecule include at least one segment of 18 ormore contiguous nucleotides designed to suppress expression of a targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof. The contiguous nucleotides of the segmentnumber at least 18, e.g., between 18-24, or between 18-28, or between20-30, or between 20-50, or between 20-100, or between 50-100, orbetween 50-500, or between 100-250, or between 100-500, or between200-1000, or between 500-2000, or even greater. In some embodiments, thecontiguous nucleotides number more than 18, e.g., 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., about 35, about40, about 45, about 50, about 55, about 60, about 65, about 70, about75, about 80, about 85, about 90, about 95, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 260, about 270, about 280, about 290, about 300, about 350,about 400, about 450, about 500, or greater than 500 contiguousnucleotides. In particular embodiments, the RNA molecule comprises atleast one segment of at least 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of equivalent length of a DNA or targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof. In particular embodiments, the RNA is adouble-stranded nucleic acid (e.g., dsRNA) with one strand comprising atleast one segment of at least 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of equivalent length of a DNA or targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof; expressed as base-pairs, such adouble-stranded nucleic acid comprises at least one segment of at least21 contiguous, perfectly matched base-pairs which correspond to afragment of equivalent length of a DNA or target gene having a sequenceselected from the Target Gene Sequences Group or the DNA complementthereof. In particular embodiments, each segment contained in the RNAmolecule is of a length greater than that which is typical of naturallyoccurring regulatory small RNAs. In some embodiments, each segment is atleast about 30 contiguous nucleotides (or base-pairs) in length. In someembodiments, the total length of the RNA molecule, or the length of eachsegment contained in the RNA molecule, is less than the total length ofthe sequence of interest (DNA or target gene having a sequence selectedfrom the group consisting of the Target Gene Sequences Group). In someembodiments, the total length of the RNA molecule is between about 50 toabout 500 nucleotides (for single-stranded polynucleotides) orbase-pairs (for double-stranded polynucleotides). In some embodiments,the RNA molecule is a dsRNA of between about 100 to about 500base-pairs, such as a dsRNA of the length of any of the dsRNA triggersdisclosed in Tables 3, 5, 8, 9, and 10. In some embodiments, theinsecticidal composition consists essentially of an insecticidallyeffective amount of a double-stranded RNA molecule with one strandhaving a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, orconsists essentially an insecticidally effective amount of an RNAhairpin encoded by a sequence selected from the group consisting of SEQID NOs:1105-1109. In some embodiments, the insecticidal compositionconsists essentially of an insecticidally effective amount of adouble-stranded RNA molecule with one strand having a sequence selectedfrom the group consisting of the Trigger Sequences Group.

The RNA molecule is generally designed to suppress one or more genes(“target genes”). Such target genes can include coding or non-codingsequence or both. In specific embodiments, the RNA molecule is designedto suppress one or more target genes, where each target gene has a DNAsequence selected from the group consisting of the Target Gene SequencesGroup. In various embodiments, the RNA molecule is designed to suppressone or more genes, where each gene has a sequence selected from thegroup consisting of the Target Gene Sequences Group, and can be designedto suppress multiple genes from this group, or to target differentregions of one or more of these genes. Embodiments of the RNA moleculeinclude at least one segment of 18 or more contiguous nucleotides havinga sequence designed to suppress one or more genes, where each gene has asequence selected from the group consisting of the Target Gene SequencesGroup. In an embodiment, the RNA molecule comprises multiple sections orsegments each of which comprises at least one segment of 21 contiguousnucleotides with a sequence of 100% complementarity to a fragment ofequivalent length of a DNA having a sequence selected from the TargetGene Sequences Group or the DNA complement thereof. In such cases, eachsection can be identical or different in size or in sequence. Forexample, in one embodiment the RNA molecule comprises multiple sectionsin tandem or repetitive arrangements, wherein each section comprises atleast one segment of 21 contiguous nucleotides with a sequence of 100%complementarity to a fragment of equivalent length of a DNA having asequence selected from the Target Gene Sequences Group or the DNAcomplement thereof; the segments can be from different regions of thetarget gene, e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments.

The RNA molecule can have a total length that is greater than 18contiguous nucleotides, and can include nucleotides in addition to thesegment of at least one segment of 18 or more contiguous nucleotideshaving the sequence of about 95% to about 100% complementarity to afragment of equivalent length of a DNA having a sequence selected fromthe Target Gene Sequences Group or the DNA complement thereof. In otherwords, the total length of the RNA molecule can be greater than thelength of the segment which is designed to suppress one or more targetgenes, where each target gene has a DNA sequence selected from the groupconsisting of the Target Gene Sequences Group. For example, the RNAmolecule can have nucleotides flanking the “active” segment of at leastone segment of 18 or more contiguous nucleotides that suppresses thetarget gene, or include “spacer” nucleotides between active segments, orcan have additional nucleotides at the 5′ end, or at the 3′ end, or atboth the 5′ and 3′ ends. In an embodiment, the RNA molecule comprisesadditional nucleotides that are not specifically related (having asequence not complementary or identical to) to the DNA or target genehaving a sequence selected from the Target Gene Sequences Group or theDNA complement thereof, e.g., nucleotides that provide stabilizingsecondary structure or for convenience in cloning or manufacturing. Inan embodiment, the RNA molecule comprises additional nucleotides locatedimmediately adjacent to one or more segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% complementarityto a fragment of equivalent length of a DNA or target gene having asequence selected from the group consisting of the Target Gene SequencesGroup. In an embodiment, the RNA molecule comprises one such segment,with an additional 5′ G or an additional 3′ C or both, adjacent to thesegment. In another embodiment, the RNA molecule is a double-strandedRNA comprising additional nucleotides to form an overhang, for example,a dsRNA comprising 2 deoxyribonucleotides to form a 3′ overhang. Thus invarious embodiments, the nucleotide sequence of the entire RNA moleculeis not 100% identical or complementary to a fragment of contiguousnucleotides in the DNA or target gene having a sequence selected fromthe group consisting of the Target Gene Sequences Group. For example, insome embodiments the RNA molecule comprises at least two segments of 21contiguous nucleotides with a sequence of 100% identity with a fragmentof a DNA having a sequence selected from the Target Gene SequencesGroup, or the DNA complement thereof, wherein (1) the at least twosegments are separated by one or more spacer nucleotides, or (2) the atleast two segments are arranged in an order different from that in whichthe corresponding fragments occur in the DNA having a sequence selectedfrom the Target Gene Sequences Group, or the DNA complement thereof.

The RNA molecule can be single-stranded (ss) or double-stranded (ds) ora combination of both. Embodiments of the RNA molecule include sensesingle-stranded RNA (ssRNA), anti-sense ssRNA, or double-stranded RNA(dsRNA), or a combination of any of these. The RNA can includecomponents other than standard ribonucleotides, e.g., an embodiment isan RNA that comprises terminal deoxyribonucleotides. In variousembodiments the RNA molecule consists of naturally occurringribonucleotides. In certain embodiments, the RNA molecule is acombination of ribonucleotides and deoxyribonucleotides, for example,synthetic RNA molecule consisting mainly of ribonucleotides but with oneor more terminal deoxyribonucleotides or one or more terminal. Incertain embodiments, the RNA molecule comprises non-canonicalnucleotides such as inosine, thiouridine, or pseudouridine. In certainembodiments, the RNA molecule comprises chemically modified nucleotides.

The RNA molecule is provided by suitable means known to one in the art.Embodiments include those wherein the RNA molecule is synthesized invitro, produced by expression in a microorganism or in cell culture(such as plant or insect cells grown in culture), produced by expressionin a plant cell, or produced by microbial fermentation.

In some embodiments the RNA molecule comprises other RNA elements, suchas RNA aptamers or ribozymes, additional non-coding RNA (e.g.,additional suppression elements), or one or more recognition sites forbinding and cleavage by a small RNA (e.g., by a miRNA or an siRNA thatis expressed only in a particular cell or tissue).

The insecticidal composition can be provided for topical application toa surface of a plant or of a plant seed, or for topical application toany substrate needing protection from a Leptinotarsa speciesinfestation. Likewise, the insecticidal composition can be provided fortopical application to a Leptinotarsa species, or in a composition foringestion by a Leptinotarsa species. In various embodiments, theinsecticidal composition is provided in the form of at least oneselected from the group consisting of a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix. Suitable binders, inert carriers, surfactants, andthe like can included in the insecticidal composition, as is known toone skilled in formulation of pesticides and seed treatments. While theinsecticidal composition consists essentially of an RNA molecule, insome embodiments the insecticidal composition further comprises at leastone non-insecticidal agent selected from the group consisting of acarrier agent, a salt, a surfactant, a cationic lipid (such as thatdisclosed in Example 18 of U.S. patent application publication2011/0296556, incorporated by reference herein), an organosilicone, anorganosilicone surfactant, a polynucleotide herbicidal molecule, anon-polynucleotide herbicidal molecule, and a safener. In one embodimentthe composition containing the recombinant RNA molecule furthercomprises a nonionic organosilicone surfactant such as SILWET® brandsurfactants, e.g., SILWET L-77® brand surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, currentlyavailable from Momentive Performance Materials, Albany, N.Y.Furthermore, the insecticidal composition can be used in combinationwith, subsequently to, or preceding, treatment with a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide (e.g., at least one pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein). Related compositions include combinations of theRNA molecule with a polynucleotide herbicidal molecule, anon-polynucleotide herbicidal molecule, a non-polynucleotide pesticide.

The insecticidal composition can be provided for dietary uptake by aLeptinotarsa species by applying the composition to a plant or surfacesubject to infestation by the Leptinotarsa species, for example byspraying, dusting, or coating the plant, or by application of a soildrench, or by providing in an artificial diet. The insecticidalcomposition can be provided for dietary uptake by a Leptinotarsa speciesin an artificial diet formulated to meet the particular nutritionalrequirements for maintaining the Leptinotarsa species, wherein theartificial diet is supplemented with some amount of the recombinant RNAmolecule obtained from a separate source such as in vitro synthesis orpurified from a microbial fermentation or other biological source; thisembodiment can be useful, e.g., for determining the timing and amountsof effective treatment regimes. The insecticidal composition can beprovided for dietary uptake by the Leptinotarsa species in the form of aseed treatment.

Methods of Providing Plants Having Improved Resistance to LeptinotarsaSpecies Infestations, and the Plants, Plant Parts, and Seeds ThusProvided

Several embodiments relate to a method of providing a plant havingimproved resistance to a Leptinotarsa species infestation comprisingproviding to the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that is essentiallyidentical or complementary to a fragment of a target gene selected fromthe group consisting of the genes identified in the Target GeneSequences Group. In an embodiment, this invention provides a method ofproviding a plant having improved resistance to a Leptinotarsa speciesinfestation comprising providing to the plant at least onepolynucleotide comprising at least one segment that is identical orcomplementary to at least 21 contiguous nucleotides of a target gene oran RNA transcribed from the target gene, wherein the target gene isselected from the genes identified in the Target Gene Sequences Group oran RNA transcribed from the target gene. Embodiments of these targetgenes are identified by name in Tables 1, 2 and 4 and include geneshaving a sequence selected from the group consisting of the Target GeneSequences Group, as well as related genes, including orthologues fromrelated insect species, for example related genes from otherLeptinotarsa species, Tribolium species, or other related genera.Examples of such related target genes include the Tribolium castaneumgenes listed in Table 1. In some embodiments, the polynucleotide is adouble-stranded RNA. In some embodiments, the polynucleotide (e.g.,double-stranded RNA) is chemically synthesized or is produced byexpression in a microorganism or by expression in a plant cell. In someembodiments the polynucleotide comprises at least one segment of 18 ormore contiguous nucleotides that is essentially identical orcomplementary to a sequence selected from the group consisting of theTarget Gene Sequences Group. In some embodiments the polynucleotide is adsRNA with a strand having a sequence selected from the group consistingof: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114, or the complementthereof, or wherein the polynucleotide is encoded by a sequence selectedfrom the group consisting of SEQ ID NOs:1105-1109. In some embodimentsthe polynucleotide comprises a dsRNA with a strand having a sequenceselected from the Trigger Sequences Group.

In one embodiment the method comprises topically applying to the plant acomposition comprising at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of a DNAof a target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group, whereby the plant treatedwith the polynucleotide composition exhibits improved resistance to aLeptinotarsa species infestation, relative to an untreated plant. By“topical application” is meant application to the surface or exterior ofan object, such as the surface or exterior of a plant, such asapplication to the surfaces of a plant part such as a leaf, stem,flower, fruit, shoot, root, seed, tuber, flowers, anthers, or pollen, orapplication to an entire plant, or to the above-ground or below-groundportions of a plant. Topical application can be carried out onnon-living surfaces, such as application to soil, or to a surface ormatrix by which a Leptinotarsa insect can come in contact with thepolynucleotide. In various embodiments of the method, thepolynucleotide-containing composition is topically applied to the plantin a suitable form, e.g., as a solid, liquid (including homogeneousmixtures such as solutions and non-homogeneous mixtures such assuspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or as a seed treatment. In some embodiments of themethod, the polynucleotide-containing composition is topically appliedto above-ground parts of the plant, e.g., sprayed or dusted onto leaves,stems, and flowering parts of the plant. Embodiments of the methodinclude topical application of a foliar spray (e.g., spraying a liquidpolynucleotide-containing composition on leaves of a solanaceous plant)or a foliar dust (e.g., dusting a solanaceous plant with apolynucleotide-containing composition in the form of a powder or oncarrier particulates). In other embodiments, thepolynucleotide-containing composition is topically applied tobelow-ground parts of the plant, such as to the roots, e.g., by means ofa soil drench. In other embodiments, the polynucleotide-containingcomposition is topically applied to a seed that is grown into the plant.The topical application can be in the form of topical treatment offruits of solanaceous plants or seeds from fruits of solanaceous plants,or in the form of topical treatment of “seed potato” tubers or pieces oftuber (e.g., by soaking, coating, or dusting the seed potato). Suitablebinders, inert carriers, surfactants, and the like can optionally beincluded in the polynucleotide-containing composition, as is known toone skilled in formulation of pesticides and seed treatments. In someembodiments, the polynucleotide-containing composition is at least onetopically implantable formulation selected from the group consisting ofa particulate, pellet, or capsule topically implanted in the plant; insuch embodiments the method comprises topically implanting in the plantthe topically implantable formulation. In some embodiments, thepolynucleotide-containing composition is at least one in-furrowformulation selected from the group consisting of a powder, granule,pellet, capsule, spray, or drench, or any other forms suited fortopically applying to a furrow; in such embodiments, the method includesan in-furrow treatment with the in-furrow formulation. In one embodimentthe polynucleotide-containing composition can be ingested or otherwiseabsorbed internally by the Leptinotarsa species. For example, thepolynucleotide-containing composition can be in the form of bait. Insome embodiments, the polynucleotide-containing composition furthercomprises one or more components selected from the group consisting of acarrier agent, a surfactant, a cationic lipid (such as that disclosed inExample 18 of U.S. patent application publication 2011/0296556,incorporated by reference herein), an organosilicone, an organosiliconesurfactant, a polynucleotide herbicidal molecule, a non-polynucleotideherbicidal molecule, a non-polynucleotide pesticide, a safener, and aninsect growth regulator. In one embodiment the composition furthercomprises a nonionic organosilicone surfactant such as SILWET® brandsurfactants, e.g., SILWET L-77® brand surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, currentlyavailable from Momentive Performance Materials, Albany, N.Y. In someembodiments, the topically applied composition further comprises atleast one pesticidal agent selected from the group consisting of apatatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein. Alternatively suchadditional components or pesticidal agents can be provided separately,e.g., by separate topical application or by transgenic expression in theplant. Alternatively the plant is topically treated with thepolynucleotide-containing composition as well as with a separate(preceding, following, or concurrent) application of a substance thatimproves the efficacy of the polynucleotide-containing composition. Forexample, a plant can be sprayed with a first topical application of asolution containing a nonionic organosilicone surfactant such as SILWET®brand surfactants, e.g., SILWET L-77® brand surfactant, followed by asecond topical application of the polynucleotide-containing composition,or vice-versa.

It is anticipated that the combination of certain polynucleotides (e.g.,the polynucleotide triggers described in the working Examples) with oneor more non-polynucleotide pesticidal agents will result in a synergeticimprovement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with thepolynucleotide alone or the non-polynucleotide pesticidal agent alone.In an embodiment, a composition containing one or more polynucleotidesand one or more non-polynucleotide pesticidal agent selected from thegroup consisting of a patatin, a plant lectin, a phytoecdysteroid, aBacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidalprotein, a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein, isfound to effect synergistically improved prevention or control ofLeptinotarsa species infestations when topically applied to a plant.

In some embodiments, the method comprises topically applying to theplant a composition comprising at least one polynucleotide comprising atleast one segment of 18 or more contiguous nucleotides that areessentially identical or complementary to a fragment of equivalentlength of a DNA of a target gene selected from the group consisting ofthe genes identified in the Target Gene Sequences Group. Thepolynucleotide topically applied to the plant can be single-stranded(ss) or double-stranded (ds).

The polynucleotide topically applied to the plant is provided bysuitable means known to one in the art. Embodiments include thosewherein the polynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation.

In many embodiments the polynucleotide topically applied to the plant isprovided as an isolated DNA or RNA. In some embodiments, thepolynucleotide topically applied to the plant is not part of anexpression construct and is lacking additional elements such as apromoter or terminator sequences. Such polynucleotides can be relativelyshort, such as single- or double-stranded polynucleotides of betweenabout 18 to about 300 or between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or between about 18 to about 300 orbetween about 50 to about 500 base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. Such recombinantexpression constructs or vectors can be designed to include additionalelements, such as expression cassettes for expressing a gene of interest(e.g., an insecticidal protein).

The polynucleotide topically applied to the plant has at least onesegment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of a DNAof a target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group, or that have a sequenceof about 95% to about 100% identity with or complementarity to afragment of equivalent length of a DNA of a target gene selected fromthe group consisting of the genes identified in the Target GeneSequences Group. In an embodiment the polynucleotide topically appliedto the plant comprises at least one segment of 18 or more contiguousnucleotides that are essentially identical or complementary to afragment of equivalent length of a DNA of a target gene selected fromthe group consisting of the genes identified in the Target GeneSequences Group. In some embodiments, the contiguous nucleotides have asequence of about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identity with or complementarity to the fragment ofequivalent length of a DNA of a target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.In some embodiments the contiguous nucleotides are exactly (100%)identical or complementary to a fragment of equivalent length of a DNAof a target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group. In some embodiments, thepolynucleotide has an overall sequence of about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identity or complementaritywith a fragment of a DNA of a target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.

The polynucleotide topically applied to the plant is generally designedto suppress one or more genes (“target genes”). In specific embodiments,the polynucleotide is designed to suppress one or more target genesselected from the group consisting of the genes identified in the TargetGene Sequences Group. Embodiments of the genes identified in the TargetGene Sequences Group include, but are not limited to, the cDNA sequencesselected from the group consisting of the Target Gene Sequences Group.In various embodiments, the polynucleotide topically applied to theplant is designed to suppress one or more genes, where each gene isselected from the group consisting of the genes identified in the TargetGene Sequences Group, and can be designed to suppress multiple genesfrom this group, or to target different regions of one or more of thesegenes. In an embodiment, the polynucleotide topically applied to theplant comprises multiple sections or segments each of which comprises atleast one segment of 18 or more contiguous nucleotides with a sequenceof about 95% to about 100% identity or complementarity with a fragmentof equivalent length of a DNA of a target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.In such cases, each section can be identical or different in size or insequence, and can be sense or anti-sense relative to the target gene.For example, in one embodiment the polynucleotide topically applied tothe plant can include multiple sections in tandem or repetitivearrangements, wherein each section comprises at least one segment of 21contiguous nucleotides with a sequence of 100% identity or 100%complementarity with a fragment of equivalent length of a DNA of atarget gene selected from the group consisting of the genes identifiedin the Target Gene Sequences Group; the segments can be from differentregions of the target gene, e.g., the segments can correspond todifferent exon regions of a cDNA with a sequence selected from the groupconsisting of the Target Gene Sequences Group, and “spacer” nucleotideswhich do not correspond to a target gene can optionally be used inbetween or adjacent to the segments.

The total length of the polynucleotide topically applied to the plantcan be greater than 18 contiguous nucleotides, and can includenucleotides in addition to the at least one segment of contiguousnucleotides having the sequence essentially identical or complementaryto a fragment of equivalent length of a DNA of a target gene selectedfrom the group consisting of the genes identified in the Target GeneSequences Group. In other words, the total length of the polynucleotidetopically applied to the plant can be greater than the length of thesection or segment of the polynucleotide designed to suppress one ormore target genes, where each target gene is selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.For example, the polynucleotide topically applied to the plant can havenucleotides flanking the “active” segment of at least one segment of 18or more contiguous nucleotides that suppresses the target gene, orinclude “spacer” nucleotides between active segments, or can haveadditional nucleotides at the 5′ end, or at the 3′ end, or at both the5′ and 3′ ends. In an embodiment, the polynucleotide topically appliedto the plant comprises additional nucleotides that are not specificallyrelated (having a sequence not complementary or identical to) to thetarget gene is selected from the group consisting of the genesidentified in the Target Gene Sequences Group, e.g., nucleotides thatprovide stabilizing secondary structure or for convenience in cloning ormanufacturing. In an embodiment, the polynucleotide topically applied tothe plant comprises additional nucleotides located immediately adjacentto one or more segment of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with or complementarity tothe target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group. In an embodiment, thepolynucleotide topically applied to the plant comprises one suchsegment, with an additional 5′ G or an additional 3′ C or both, adjacentto the segment. In another embodiment, the polynucleotide topicallyapplied to the plant is a double-stranded RNA comprising additionalnucleotides to form an overhang, for example, a dsRNA comprising 2deoxyribonucleotides to form a 3′ overhang. Thus in various embodiments,the nucleotide sequence of the entire polynucleotide topically appliedto the plant is not 100% identical or complementary to a fragment ofcontiguous nucleotides in the target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.For example, in some embodiments the polynucleotide topically applied tothe plant comprises at least two segments each of 21 contiguousnucleotides with a sequence of 100% identity with a fragment of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group, wherein (1) the at least two segments areseparated by one or more spacer nucleotides, or (2) the at least twosegments are arranged in an order different from that in which thecorresponding fragments occur in the target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.

In a related aspect, this invention is directed to the plant havingimproved resistance to a Leptinotarsa species infestation, provided bythis method which comprises topically applying to the plant acomposition comprising at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of a DNAof a target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group, whereby the plant treatedwith the polynucleotide composition exhibits improved resistance to aLeptinotarsa species infestation, relative to an untreated plant. In yetanother aspect, this invention is directed to seed (especiallytransgenic progeny seed) produced by the plant having improvedresistance to a Leptinotarsa species infestation, as provided by thismethod. Also contemplated is a commodity product produced by the planthaving improved resistance to a Leptinotarsa species infestation, asprovided by this method, and a commodity product produced from thetransgenic progeny seed of such a plant.

In another embodiment the method comprises expressing in the plant atleast one polynucleotide comprising at least one segment of 18 or morecontiguous nucleotides that are essentially identical or complementaryto a fragment of equivalent length of a target gene selected from thegroup consisting of the genes identified in the Target Gene SequencesGroup, whereby the plant expressing the polynucleotide exhibits improvedresistance to a Leptinotarsa species infestation, relative to an plantnot expressing the polynucleotide. In an embodiment the method comprisesexpressing in the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides with a sequence ofabout 95% to about 100% identity or complementarity with a fragment ofequivalent length of a DNA of a target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.Embodiments of these target genes are identified by name in Tables 1, 2and 4 and include genes having a sequence selected from the groupconsisting of the Target Gene Sequences Group, as well as related genes,including orthologues from related insect species, for example relatedgenes from other Leptinotarsa species, Tribolium species, or otherrelated genera. Examples of such related target genes include theTribolium castaneum genes listed in Table 1. By “expressing apolynucleotide in the plant” is generally meant “expressing an RNAtranscript in the plant”. However, the polynucleotide expressed in theplant can also be DNA, e.g., a DNA produced in the plant during genomereplication.

The method comprises expressing at least one polynucleotide in a plant,wherein the polynucleotide comprises at least one segment of 18 or morecontiguous nucleotides that is essentially identical or complementary toa fragment of a target gene selected from the group consisting of thegenes identified in the Target Gene Sequences Group. In someembodiments, a first polynucleotide is provided to a plant in the formof DNA (e.g., in the form of an isolated DNA molecule, or as anexpression construct, or as a transformation vector), and thepolynucleotide expressed in the plant is a second polynucleotide (e.g.,the RNA transcript of the first polynucleotide) in the plant. In anembodiment, the polynucleotide is expressed in the plant by transgenicexpression, i.e., by stably integrating the polynucleotide into theplant's genome from where it can be expressed in a cell or cells of theplant. In an embodiment, a first polynucleotide (e.g., a recombinant DNAconstruct comprising a promoter operably linked to DNA comprising atleast one segment of 18 or more contiguous nucleotides that isessentially identical or complementary to a fragment of target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group is stably integrated into the plant's genome fromwhere secondarily produced polynucleotides (e.g., an RNA transcriptcomprising the transcript of the segment of 18 or more contiguousnucleotides that is essentially identical or complementary to a fragmentof target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group) are expressed in a cellor cells of the plant. Methods of providing stably transformed plantsare provided in the section headed “Making and Using Transgenic PlantCells and Transgenic Plants”.

In another embodiment the polynucleotide expressed in the plant isexpressed by transient expression (i.e., expression not resulting fromstable integration of a sequence into the plant's genome). In suchembodiments the method can include a step of introducing apolynucleotide (e.g., dsRNA or dsDNA) into the plant by routinetechniques known in the art. For example, transient expression can beaccomplished by infiltration of a polynucleotide solution using aneedle-less syringe into a leaf of a plant.

In some embodiments where the polynucleotide expressed in the plant isexpressed by transient expression, a first polynucleotide is provided toa plant in the form of RNA or DNA or both RNA and DNA, and a secondarilyproduced second polynucleotide is transiently expressed in the plant. Insome embodiments, the first polynucleotide is one or more selected from:(a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNAmolecule that self-hybridizes to form a double-stranded RNA molecule,(c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNAmolecule (ssDNA), (e) a single-stranded DNA molecule thatself-hybridizes to form a double-stranded DNA molecule, (f) asingle-stranded DNA molecule comprising a modified Pol III gene that istranscribed to an RNA molecule, (g) a double-stranded DNA molecule(dsDNA), (h) a double-stranded DNA molecule comprising a modified PolIII gene that is transcribed to an RNA molecule, and (i) adouble-stranded, hybridized RNA/DNA molecule, or combinations thereof.In specific embodiments, a first polynucleotide is introduced into theplant by topical application to the plant of a polynucleotide-containingcomposition in a suitable form, e.g., as a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or in the form of a treatment of a solanaceous plantseed or treatment of a seed potato. Suitable binders, inert carriers,surfactants, and the like can optionally be included in the composition,as is known to one skilled in formulation of pesticides and seedtreatments. In such embodiments, the polynucleotide-containingcomposition can further include one or more components selected from thegroup consisting of a carrier agent, a surfactant, a cationic lipid(such as that disclosed in Example 18 of U.S. patent applicationpublication 2011/0296556, incorporated by reference herein), anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator;in one embodiment the composition further comprises a nonionicorganosilicone surfactant such as SILWET® brand surfactants, e.g.,SILWET L-77® brand surfactant having CAS Number 27306-78-1 and EPANumber: CAL.REG.NO. 5905-50073-AA, currently available from MomentivePerformance Materials, Albany, N.Y. In some embodiments, the topicallyapplied composition further comprises at least one pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein. Alternatively such additional components orpesticidal agents can be provided separately, e.g., by separate topicalapplication or by transgenic expression in the plant. Alternatively theplant is topically treated with the polynucleotide-containingcomposition as well as with a separate (preceding, following, orconcurrent) application of a substance that improves the efficacy of thepolynucleotide-containing composition. For example, a plant can besprayed with a first topical application of a solution containing anonionic organosilicone surfactant such as SILWET® brand surfactants,e.g., SILWET L-77® brand surfactant, followed by a second topicalapplication of the polynucleotide-containing composition, or vice-versa.

It is anticipated that the combination of certain polynucleotides of usein this method (e.g., the polynucleotide triggers described in theworking Examples) with one or more non-polynucleotide pesticidal agentswill result in a synergetic improvement in prevention or control ofLeptinotarsa species infestations, when compared to the effect obtainedwith the polynucleotide alone or the non-polynucleotide pesticidal agentalone. In an embodiment, a transgenic plant expressing at least onepolynucleotide comprising at least one segment of 18 or more contiguousnucleotides that is essentially identical or complementary to a fragmentof target gene selected from the group consisting of the genesidentified in Table 1 (e.g., the polynucleotide triggers described inthe working Examples) and one or more genes encoding anon-polynucleotide pesticidal agent selected from the group consistingof a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein, isfound to exhibit synergistically improved resistance to Leptinotarsaspecies infestations.

In some embodiments where the polynucleotide expressed in the plant isexpressed by transient expression, a first polynucleotide is provided toa plant in the form of RNA or DNA or both RNA and DNA, and a secondarilyproduced second polynucleotide is transiently expressed in the plant;the site of application of the first polynucleotide need not be the samesite where the second polynucleotide is transiently expressed. Forexample, a first polynucleotide can be provided to a plant by topicalapplication onto a leaf, or by injection into a stem, and the secondpolynucleotide can be transiently expressed elsewhere in the plant,e.g., in the roots or throughout the plant. In some embodiments of themethod, a composition comprising at least one polynucleotide istopically applied to above-ground parts of the plant, e.g., sprayed ordusted onto leaves, stems, and flowering parts of the plant. In otherembodiments, a composition comprising at least one polynucleotide istopically applied to below-ground parts of the plant, such as to theroots, e.g., by means of a soil drench. In other embodiments, acomposition comprising at least one polynucleotide is topically appliedto a seed (or, in the case of potatoes, topically applied to a seedpotato) that is grown into the plant having improved resistance to aLeptinotarsa species infestation.

In some embodiments the polynucleotide expressed in the plant is RNA,which can be single-stranded (ss) or double-stranded (ds) RNA or acombination of both.

In some embodiments a first polynucleotide (DNA or RNA or both) isprovided to a plant and a second polynucleotide having a sequencecorresponding (identical or complementary) to the first polynucleotideis subsequently expressed in the plant. In such embodiments thepolynucleotide expressed in the plant is an RNA transcript which can bessRNA or dsRNA or a combination of both. In some embodiments where thepolynucleotide is expressed by transient expression, a firstpolynucleotide is provided to a plant in the form of RNA or DNA or bothRNA and DNA, and a secondarily produced second polynucleotide istransiently expressed in the plant; in such embodiments, the firstpolynucleotide one or more selected from: (a) a single-stranded RNAmolecule (ssRNA), (b) a single-stranded RNA molecule thatself-hybridizes to form a double-stranded RNA molecule, (c) adouble-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule(ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to forma double-stranded DNA molecule, (f) a single-stranded DNA moleculecomprising a modified Pol III gene that is transcribed to an RNAmolecule, (g) a double-stranded DNA molecule (dsDNA), (h) adouble-stranded DNA molecule comprising a modified Pol III gene that istranscribed to an RNA molecule, and (i) a double-stranded, hybridizedRNA/DNA molecule, or combinations thereof. In such embodiments where thepolynucleotide is expressed by transient expression the firstpolynucleotide can consist of naturally occurring nucleotides, such asthose which occur in DNA and RNA. In such embodiments where thepolynucleotide is expressed by transient expression the firstpolynucleotide can be chemically modified, or comprises chemicallymodified nucleotides. The first polynucleotide is provided by suitablemeans known to one in the art. Embodiments include those wherein thefirst polynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation. Thefirst polynucleotide can be provided as an RNA or DNA fragment.Alternatively the first polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector; such recombinantexpression constructs or vectors can be designed to include additionalelements, such as expression cassettes for expressing a gene of interest(e.g., an insecticidal protein).

In some embodiments the polynucleotide expressed in the plant is an RNAmolecule and can be relatively short, such as single- or double-strandedRNAs of between about 18 to about 300 or between about 50 to about 500nucleotides (for single-stranded RNAs) or between about 18 to about 300or between about 50 to about 500 base-pairs (for double-stranded RNAs).Alternatively the polynucleotide can be provided in more complexconstructs, e.g., as part of a recombinant expression construct, orincluded in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. In some embodimentssuch recombinant expression constructs or vectors are designed toinclude additional elements, such as expression cassettes for expressinga gene of interest (e.g., an insecticidal protein).

The polynucleotide expressed in the plant has at least one segment of 18or more contiguous nucleotides with a sequence of about 95% to about100% identity with or complementarity to a fragment of equivalent lengthof a target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group. In an embodiment thepolynucleotide expressed in the plant comprises at least one segment of18 or more contiguous nucleotides that are essentially identical orcomplementary to a fragment of equivalent length of a target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group. In some embodiments, the contiguous nucleotideshave a sequence of about 95%, about 96%, about 97%, about 98%, about99%, or about 100% identity with or complementarity to a fragment ofequivalent length of a target gene selected from the group consisting ofthe genes identified in the Target Gene Sequences Group. In someembodiments the contiguous nucleotides are exactly (100%) identical orcomplementary to a fragment of equivalent length of a target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group. In some embodiments, the polynucleotide expressedin the plant has an overall sequence of about 95%, about 96%, about 97%,about 98%, about 99%, or about 100% identity with or complementarity toa fragment of a target gene selected from the group consisting of thegenes identified in the Target Gene Sequences Group.

The polynucleotide expressed in the plant is generally designed tosuppress one or more genes (“target genes”). Such target genes caninclude coding or non-coding sequence or both. In specific embodiments,the polynucleotide expressed in the plant is designed to suppress one ormore target genes selected from the group consisting of the genesidentified in the Target Gene Sequences Group. In various embodiments,the polynucleotide expressed in the plant is designed to suppress one ormore target genes selected from the group consisting of the genesidentified in the Target Gene Sequences Group, and can be designed tosuppress multiple genes from this group, or to target different regionsof one or more of these genes. In an embodiment, the polynucleotideexpressed in the plant comprises multiple sections or segments each ofwhich comprises at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with orcomplementarity to a fragment of a target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.In such cases, each section can be identical or different in size or insequence, and can be sense or anti-sense relative to the target gene.For example, in one embodiment the polynucleotide expressed in the plantcan include multiple sections in tandem or repetitive arrangements,wherein each section comprises at least one segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity with or complementarity to a fragment of a target gene selectedfrom the group consisting of the genes identified in the Target GeneSequences Group; the segments can be from different regions of thetarget gene, e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments.

The total length of the polynucleotide expressed in the plant can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with or complementarity to a fragment of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group. In other words, the total length of thepolynucleotide expressed in the plant can be greater than the length ofthe section or segment of the polynucleotide designed to suppress one ormore target genes selected from the group consisting of the genesidentified in the Target Gene Sequences Group. For example, thepolynucleotide expressed in the plant can have nucleotides flanking the“active” segment of at least one segment of 18 or more contiguousnucleotides that suppresses the target gene, or include “spacer”nucleotides between active segments, or can have additional nucleotidesat the 5′ end, or at the 3′ end, or at both the 5′ and 3′ ends. In anembodiment, the polynucleotide expressed in the plant comprisesadditional nucleotides that are not specifically related (having asequence not complementary or identical to) to the target gene selectedfrom the group consisting of the genes identified in the Target GeneSequences Group, e.g., nucleotides that provide stabilizing secondarystructure or for convenience in cloning or manufacturing. In anembodiment, the polynucleotide expressed in the plant comprisesadditional nucleotides located immediately adjacent to one or moresegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with or complementarity to a fragment ofequivalent length of a target gene selected from the group consisting ofthe genes identified in the Target Gene Sequences Group. In anembodiment, the polynucleotide expressed in the plant comprises one suchsegment, with an additional 5′ G or an additional 3′ C or both, adjacentto the segment. In another embodiment, the polynucleotide expressed inthe plant is a double-stranded RNA comprising additional nucleotides toform an overhang, for example, a dsRNA comprising 2 deoxyribonucleotidesto form a 3′ overhang. Thus in various embodiments, the nucleotidesequence of the entire polynucleotide expressed in the plant is not 100%identical or complementary to a fragment of contiguous nucleotides inthe target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group. For example, in someembodiments the polynucleotide expressed in the plant comprises at leasttwo segments of 21 contiguous nucleotides with a sequence of 100%identity with or 100% complementarity to a fragment of a target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group, wherein (1) the at least two segments areseparated by one or more spacer nucleotides, or (2) the at least twosegments are arranged in an order different from that in which thecorresponding fragments occur in the target gene selected from the groupconsisting of the genes identified in the Target Gene Sequences Group.

In a related aspect, this invention is directed to the plant havingimproved resistance to a Leptinotarsa species infestation, provided byexpressing in the plant at least one polynucleotide comprising at leastone segment of 18 or more contiguous nucleotides that are essentiallyidentical or complementary to a fragment of equivalent length of atarget gene selected from the group consisting of the genes identifiedin the Target Gene Sequences Group, whereby the resulting plant hasimproved resistance to a Leptinotarsa species infestation when comparedto a control plant in which the polynucleotide is not expressed. In arelated aspect, this invention is directed to the plant having improvedresistance to a Leptinotarsa species infestation, provided by expressingin the plant at least one polynucleotide comprising at least one segmentof 18 or more contiguous nucleotides with a sequence of about 95% toabout 100% identity with or complementarity to a fragment of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group, whereby the resulting plant has improvedresistance to a Leptinotarsa species infestation when compared to acontrol plant in which the polynucleotide is not expressed. Anembodiment is a solanaceous plant having improved resistance to aLeptinotarsa species infestation when compared to a control plant,provided by expressing in the plant an RNA having a sequence selectedfrom the group consisting of SEQ ID NOs:831-1085 and 1095. In yetanother aspect, this invention is directed to seed or propagatable parts(especially transgenic progeny seed or propagatable parts) produced bythe plant having improved resistance to a Leptinotarsa speciesinfestation, as provided by this method. Also contemplated is acommodity product produced by the plant having improved resistance to aLeptinotarsa species infestation, as provided by this method, and acommodity product produced from the transgenic progeny seed orpropagatable parts of such a plant.

Methods of Controlling Leptinotarsa Species Infestations of a Plant

Several embodiments relate to a method for controlling a Leptinotarsaspecies infestation of a plant comprising contacting the Leptinotarsaspecies with a polynucleotide comprising at least one segment of 18 ormore contiguous nucleotides that is essentially identical orcomplementary to a fragment of equivalent length of a DNA of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group. In this context “controlling” includesinducement of a physiological or behavioural change in a Leptinotarsaspecies (adult or larvae) such as, but not limited to, growth stunting,increased mortality, decrease in reproductive capacity, decrease in orcessation of feeding behavior or movement, or decrease in or cessationof metamorphosis stage development. In an embodiment, the method forcontrolling a Leptinotarsa species infestation of a plant comprisescontacting the Leptinotarsa species with a polynucleotide comprising atleast one segment that is identical or complementary to at least 21contiguous nucleotides of a target gene selected from the genesidentified in the Target Gene Sequences Group or an RNA transcribed fromthe target gene. In some embodiments, the polynucleotide is adouble-stranded RNA. In some embodiments, the polynucleotide (e.g.,double-stranded RNA) is chemically synthesized or is produced byexpression in a microorganism or by expression in a plant cell. In someembodiments, the polynucleotide is a double-stranded RNA comprising astrand comprising a sequence selected from the Trigger Sequences Group.In an embodiment, the method for controlling a Leptinotarsa speciesinfestation of a plant comprises contacting the Leptinotarsa specieswith an effective amount of a double-stranded RNA, one strand of whichis complementary to at least 21 contiguous nucleotides of a gene thatencodes a ribosomal protein, wherein RNA interference is induced andmortality occurs. Embodiments of target genes are identified by name inTables 1, 2 and 4 and include genes having a sequence selected from thegroup consisting of the Target Gene Sequences Group, as well as relatedgenes including orthologues from related insect species, for example,related genes from other Leptinotarsa species, Tribolium species, orother related coleopteran genera. Examples of such related genes includethe Tribolium castaneum genes listed in Table 1. In some embodiments thepolynucleotide comprises at least one segment of 18 or more contiguousnucleotides that is essentially identical or complementary to a fragmentof a target gene having a sequence selected from the group consisting ofthe Target Gene Sequences Group. In some embodiments the polynucleotidecomprises RNA having a sequence selected from the group consisting ofSEQ ID NOs:831-1085, 1095-1104, and 1110-1114, or the complementthereof, or is an RNA hairpin encoded by a sequence selected from thegroup consisting of SEQ ID NOs:1105-1109. In some embodiments thepolynucleotide comprises a dsRNA with a strand having a sequenceselected from the group consisting of the Trigger Sequences Group. Insome embodiments, this invention provides a method for controlling aLeptinotarsa species infestation of a plant comprising contacting theLeptinotarsa species with an effective amount of a solution comprising adouble-stranded RNA, wherein at least one strand of the double-strandedRNA is complementary to at least 21 contiguous nucleotides of a genethat encodes a ribosomal protein or an RNA transcribed from the gene,wherein the Leptinotarsa species is Leptinotarsa decemlineata, andwherein RNA interference is induced and Leptinotarsa decemlineatamortality occurs, and wherein the ribosomal protein is a ribosomal L7protein or a protein encoded by SEQ ID NO:730 or wherein thedouble-stranded RNA comprises a sequence selected from the groupconsisting of SEQ ID NO:989, 988, 1104, or 1105; in some embodiments,the solution further comprises one or more components selected from thegroup consisting of an organosilicone surfactant or a cationic lipid.

In some embodiments, the contiguous nucleotides have a sequence of about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identitywith or complementarity to a fragment of equivalent length of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group. In some embodiments the contiguousnucleotides are exactly (100%) identical or complementary to a fragmentof equivalent length of a target gene selected from the group consistingof the genes identified in the Target Gene Sequences Group. In someembodiments, the polynucleotide has an overall sequence of about 95%,about 96%, about 97%, about 98%, about 99%, or about 100% identity withor complementarity to a fragment of equivalent length of a target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group. In an embodiment, the polynucleotide comprises atleast one segment of 21 contiguous nucleotides with a sequence of 100%identity or complementarity with the corresponding fragment of a targetgene selected from the group consisting of the genes identified in theTarget Gene Sequences Group; in some embodiments, the polynucleotidecomprises “neutral” sequence (having no sequence identity orcomplementarity to the target gene) in addition to a segment of 21contiguous nucleotides with 100% identity with the correspondingfragment of the target gene, and therefore the polynucleotide as a wholeis of much lower overall sequence identity with a target gene.

The polynucleotide of use in this method is generally designed tosuppress one or more genes (“target genes”). The term “gene” refers toany portion of a nucleic acid that provides for expression of atranscript or encodes a transcript. A “gene” can include, but is notlimited to, a promoter region, 5′ untranslated regions, transcriptencoding regions that can include intronic regions, 3′ untranslatedregions, or combinations of these regions. In some embodiments, thetarget genes can include coding or non-coding sequence or both. In otherembodiments, the target gene has a sequence identical to orcomplementary to a messenger RNA, e.g., in some embodiments the targetgene is a cDNA. In specific embodiments, the polynucleotide is designedto suppress one or more target genes selected from the group consistingof the genes identified in the Target Gene Sequences Group. In variousembodiments, the polynucleotide is designed to suppress one or moretarget genes selected from the group consisting of the genes identifiedin the Target Gene Sequences Group, and can be designed to suppressmultiple target genes from this group, or to target different regions ofone or more of these target genes. In an embodiment, the polynucleotidecomprises multiple segments of 21 contiguous nucleotides with a sequenceof 100% identity with a fragment of equivalent length of a DNA or targetgene having a sequence selected from the Target Gene Sequences Group orthe DNA complement thereof. In such cases, each segment can be identicalor different in size or in sequence, and can be sense or anti-senserelative to the target gene. For example, in one embodiment thepolynucleotide comprises multiple segments in tandem or repetitivearrangements, wherein each segment comprises 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with orcomplementarity to a fragment of equivalent length of a target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group; the segments can be from different regions of thetarget gene, e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments.

The total length of the polynucleotide of use in this method can begreater than 18 contiguous nucleotides, and can include nucleotides inaddition to the contiguous nucleotides having the sequence of about 95%to about 100% identity with or complementarity to a fragment ofequivalent length of a target gene selected from the group consisting ofthe genes identified in the Target Gene Sequences Group. In other words,the total length of the polynucleotide can be greater than the length ofthe section or segment of the polynucleotide designed to suppress one ormore target genes selected from the group consisting of the genesidentified in the Target Gene Sequences Group. For example, thepolynucleotide can have nucleotides flanking the “active” segment of atleast one segment of 18 or more contiguous nucleotides that suppressesthe target gene, or include “spacer” nucleotides between activesegments, or can have additional nucleotides at the 5′ end, or at the 3′end, or at both the 5′ and 3′ ends. In an embodiment, the polynucleotidecan include additional nucleotides that are not specifically related(having a sequence not complementary or identical to) to the target geneselected from the group consisting of the genes identified in the TargetGene Sequences Group, e.g., nucleotides that provide stabilizingsecondary structure or for convenience in cloning or manufacturing. Inan embodiment, the polynucleotide can include additional nucleotideslocated immediately adjacent to one or more segment of 18 or morecontiguous nucleotides with a sequence of about 95% to about 100%identity with or complementarity to a fragment of equivalent length of atarget gene selected from the group consisting of the genes identifiedin the Target Gene Sequences Group. In an embodiment, the polynucleotidecomprises one such segment, with an additional 5′ G or an additional 3′C or both, adjacent to the segment. In another embodiment, thepolynucleotide is a double-stranded RNA comprising additionalnucleotides to form an overhang, for example, a dsRNA comprising 2deoxyribonucleotides to form a 3′ overhang. Thus in various embodiments,the nucleotide sequence of the entire polynucleotide is not 100%identical or complementary to a sequence of contiguous nucleotides inthe target gene selected from the group consisting of the genesidentified in the Target Gene Sequences Group. For example, in someembodiments the polynucleotide comprises at least two segments each of21 contiguous nucleotides with a sequence of 100% identity with afragment of equivalent length of the target gene, wherein (1) the atleast two segments are separated by one or more spacer nucleotides, or(2) the at least two segments are arranged in an order different fromthat in which the corresponding fragments occur in the DNA having asequence selected from the Target Gene Sequences Group, or the DNAcomplement thereof.

The polynucleotide of use in this method is provided by suitable meansknown to one in the art. Embodiments include those wherein thepolynucleotide is chemically synthesized (e.g., by in vitrotranscription, such as transcription using a T7 polymerase or otherpolymerase), produced by expression in a microorganism or in cellculture (such as plant or insect cells grown in culture), produced byexpression in a plant cell, or produced by microbial fermentation.

In some embodiments the polynucleotide of use in this method is providedas an isolated DNA or RNA fragment. In some embodiments thepolynucleotide of use in this method is not part of an expressionconstruct and is lacking additional elements such as a promoter orterminator sequences). Such polynucleotides can be relatively short,such as single- or double-stranded polynucleotides of between about 18to about 300 or between about 50 to about 500 nucleotides (forsingle-stranded polynucleotides) or between about 18 to about 300 orbetween about 50 to about 500 base-pairs (for double-strandedpolynucleotides). In some embodiments, the polynucleotide is a dsRNA ofbetween about 100 to about 500 base-pairs, such as a dsRNA of the lengthof any of the dsRNA triggers disclosed in Tables 3, 5, 8, 9, and 10.Embodiments include those in which the polynucleotide is a dsRNAcomprising a segment having a sequence selected from the groupconsisting of: SEQ ID NOs:831-1085, 1095-1104, and 1110-1114, or thecomplement thereof, or wherein the polynucleotide is an RNA hairpinencoded by a sequence selected from the group consisting of SEQ IDNOs:1105-1109. Alternatively the polynucleotide can be provided in morecomplex constructs, e.g., as part of a recombinant expression construct,or included in a recombinant vector, for example in a recombinant plantvirus vector or in a recombinant baculovirus vector. In some embodimentssuch recombinant expression constructs or vectors are designed toinclude additional elements, such as expression cassettes for expressinga gene of interest (e.g., an insecticidal protein).

In various embodiments of the method, the contacting comprisesapplication to a surface of the Leptinotarsa species of a suitablecomposition comprising the polynucleotide of use in this method; such acomposition can be provided, e.g., as a solid, liquid (includinghomogeneous mixtures such as solutions and non-homogeneous mixtures suchas suspensions, colloids, micelles, and emulsions), powder, suspension,emulsion, spray, encapsulated or micro-encapsulation formulation, in oron microbeads or other carrier particulates, in a film or coating, or onor within a matrix, or as a seed treatment. The contacting can be in theform of a seed treatment, or in the form of treatment of “seed potato”tubers or pieces of tuber (e.g., by soaking, coating, or dusting theseed potato). Suitable binders, inert carriers, surfactants, and thelike can optionally be included in the composition, as is known to oneskilled in formulation of pesticides and seed treatments. In someembodiments, the contacting comprises providing the polynucleotide in acomposition that further comprises one or more components selected fromthe group consisting of a carrier agent, a surfactant, a cationic lipid(such as that disclosed in Example 18 of U.S. patent applicationpublication 2011/0296556, incorporated by reference herein), anorganosilicone, an organosilicone surfactant, a polynucleotideherbicidal molecule, a non-polynucleotide herbicidal molecule, anon-polynucleotide pesticide, a safener, and an insect growth regulator.In embodiments, the contacting comprises providing the polynucleotide ina composition that further comprises at least one pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein. In one embodiment the contacting comprisesproviding the polynucleotide in a composition that can be ingested orotherwise absorbed internally by the Leptinotarsa species.

It is anticipated that the combination of certain polynucleotides of usein this method (e.g., the polynucleotide triggers described in theworking Examples) with one or more non-polynucleotide pesticidal agentswill result in a synergetic improvement in prevention or control ofLeptinotarsa species infestations, when compared to the effect obtainedwith the polynucleotide alone or the non-polynucleotide pesticidal agentalone. In an embodiment, a composition containing one or morepolynucleotides and one or more non-polynucleotide pesticidal agentselected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein, is found to effect synergistically improvedprevention or control of Leptinotarsa species infestations.

Methods of Selecting Target Genes

Another aspect of this invention provides a method of non-randomselection of target genes for RNAi-mediated silencing. In an embodiment,the method provides a subset of target genes that are present in single-or low-copy-number (non-repetitive and non-redundant) in a particulargenome. Such target genes can be genes from a plant genome or genes froman animal genome. In some embodiments, the target genes are genes of aninvertebrate pest, e.g. an invertebrate pest of a plant or aninvertebrate pest of a vertebrate. In some embodiments, the target genesare genes of an insect pest of a plant or a nematode pest of a plant. Insome embodiments, the target genes are genes of a Leptinotarsa species.Further aspects include manufacturing a polynucleotide (e.g., an ssRNAor dsRNA trigger, such as the dsRNA triggers described in the workingExamples, or a recombinant DNA construct useful for making transgenicplants) based on target genes for RNAi-mediated silencing selected byany of the methods described herein.

In an embodiment, the method comprises the step of identifying single-or low-copy-number genes in the chosen genome, or alternatively toidentify single- or low-copy-number genes in an orthologous databasefrom related organisms to predict which genes will be single/low copy inthe chosen organism. Low-copy genes, and in particular single-copygenes, are selected as targets for RNAi-mediated silencing. In oneembodiment, the identification of single- or low-copy-number genes iscarried out by sequence comparison between a set of genes from a firstspecies and a set of genes from a second species, wherein the set ofgenes from a second species have been identified as single- orlow-copy-number in the second species. In one embodiment, theidentification of single- or low-copy-number genes is carried out byapplying an algorithm performed by a computer to a set of genes from afirst species to identify a subset of single- or low-copy-number genesin the set of genes from the first species, then comparing a set ofgenes from a second species to the subset of single- or low-copy-numbergenes from the first species to identify corresponding single- orlow-copy-number genes from the second species. The single- orlow-copy-number genes from the second species are useful as target genesfor RNAi-mediated silencing; the sequences of these target genes areused for designing polynucleotides (e.g., an ssRNA or dsRNA trigger,such as the dsRNA triggers described in the working Examples, orrecombinant DNA constructs for making transgenic plants) and methods ofuse thereof for preventing or controlling infestations by the secondspecies.

Embodiments of the method include a further step of estimatingnucleotide diversity for low-/single-copy genes in a population of thechosen organism and selecting those low-/single-copy genes that furtherhave the lowest nucleotide diversity. Low-/single-copy genes thatfurther have low nucleotide diversity are selected as targets forRNAi-mediated silencing.

Embodiments of the method include a further step of comparing the ratioof synonymous (K_(s)) to nonsynonymous (K_(a)) nucleotide changes as anestimate of functional or evolutionary constraint. In an embodiment, themethod comprises the step of selecting genes where K_(s) is at leastequal to or greater than K_(a). In an embodiment, the method comprisesthe step of selecting genes where K_(a)>>K_(a).

A related aspect of this invention is a set of target genes forRNAi-mediated silencing identified from a genome by any of the geneselection methods described herein. An embodiment relates to a set oftarget genes for RNAi-mediated silencing selected from a genome byidentifying single- or low-copy-number target genes from a larger set ofgenes from that genome. One embodiment relates to a set of target genesfor RNAi-mediated silencing selected from an invertebrate genome byidentifying single- or low-copy-number target genes from a larger set ofgenes from that invertebrate genome. A specific embodiment relates to aset of target genes for RNAi-mediated silencing in a Leptinotarsaspecies selected from a Leptinotarsa genome by identifying single- orlow-copy-number target genes from a larger set of genes from thatLeptinotarsa genome. A specific embodiment relates to a set of targetgenes for RNAi-mediated silencing in a Leptinotarsa species selectedfrom a Leptinotarsa genome by identifying single- or low-copy-numbertarget genes from a larger set of genes from that Leptinotarsa genome,wherein the set of sequences is the group consisting of SEQ IDNOs:1-725, or the DNA complement thereof.

Related aspects of this invention are methods and compositions utilisingthe set of target genes consisting of SEQ ID NOs:1-725, or the DNAcomplement thereof. These include: (i) a method for controlling aLeptinotarsa species infestation of a plant comprising contacting theLeptinotarsa species with a polynucleotide comprising at least onesegment of 18 or more contiguous nucleotides with a sequence of about95% to about 100% identity with a segment of equivalent length of a DNAhaving a sequence selected from the group consisting of: SEQ IDNOs:1-725, or the DNA complement thereof; (ii) a method for controllinga Leptinotarsa species infestation of a plant comprising providing inthe diet of a Leptinotarsa species an agent comprising a polynucleotidehaving at least one segment of 18 or more contiguous nucleotides with asequence of about 95% to about 100% identity with a segment ofequivalent length of a DNA having a sequence selected from the groupconsisting of: SEQ ID NOs:1-725, or the DNA complement thereof, whereinthe agent functions upon ingestion by the Leptinotarsa species toinhibit a biological function within the Leptinotarsa species therebycontrolling infestation by the Leptinotarsa species; (iii) a method ofcausing mortality or stunting in Leptinotarsa species larvae comprisingproviding in the diet of Leptinotarsa species larvae at least onerecombinant RNA comprising at least one silencing element essentiallyidentical or essentially complementary to a target gene of theLeptinotarsa species larvae, wherein the target gene sequence isselected from the group consisting of SEQ ID NOs:1-725; (iv) a method ofproviding a plant having improved resistance to a Leptinotarsa speciesinfestation comprising topically applying to the plant a compositioncomprising at least one polynucleotide having at least one segment of 18or more contiguous nucleotides with a sequence of about 95% to about100% identity with a segment of equivalent length of a DNA having asequence selected from the group consisting of: SEQ ID NOs:1-725, or theDNA complement thereof; (v) a composition for controlling a Leptinotarsaspecies comprising at least one recombinant polynucleotide comprising atleast one segment of 18 or more contiguous nucleotides that isessentially identical or complementary to a segment of equivalent lengthof a DNA having a sequence selected from the group consisting of SEQ IDNOs:1-725; (vi) a method of providing a plant having improved resistanceto a Leptinotarsa species infestation comprising expressing in the plantat least one polynucleotide comprising at least one segment of 18 ormore contiguous nucleotides that is essentially identical orcomplementary to a segment of equivalent length of a DNA having asequence selected from the group consisting of SEQ ID NOs:1-725; (vii) arecombinant DNA construct comprising a heterologous promoter operablylinked to DNA comprising at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with asegment of equivalent length of a DNA having a sequence selected fromthe group consisting of: SEQ ID NOs:1-725, or the DNA complementthereof; and (viii) a transgenic solanaceous plant cell having in itsgenome a recombinant DNA encoding RNA that suppresses expression of atarget gene in a Leptinotarsa species that contacts or ingests the RNA,wherein the RNA comprises at least one silencing element complementaryto the target gene, and wherein the target gene sequence is a sequenceselected from the group consisting of: SEQ ID NOs:1-725, or thecomplement thereof.

Another embodiment relates to a set of target genes for RNAi-mediatedsilencing selected from a genome by estimating nucleotide diversity fora given set of genes in a population of individuals of the specieshaving that genome, and selecting those genes that have the lowestnucleotide diversity. One embodiment relates to a set of target genesfor RNAi-mediated silencing selected from an invertebrate genome byestimating nucleotide diversity for a given set of genes in a populationof individuals of the invertebrate having that genome, and selectingthose genes that have the lowest nucleotide diversity. Anotherembodiment relates to a set of target genes for RNAi-mediated silencingselected from an invertebrate genome by estimating nucleotide diversityfor low-/single-copy genes in a population of individuals of theinvertebrate having that genome, and selecting those low-/single-copygenes that further have the lowest nucleotide diversity.

Another embodiment relates to a set of target genes for RNAi-mediatedsilencing selected from a genome by comparing the ratio of synonymous(K_(s)) to nonsynonymous (K_(a)) nucleotide changes in genes of thatgenome and selecting genes where K_(s) is at least equal to or greaterthan K_(a). In an embodiment, the set of target genes for RNAi-mediatedsilencing are genes where K_(s) is at least equal to or greater thanK_(a). In an embodiment, the set of target genes for RNAi-mediatedsilencing are genes where K_(s)>>K_(a). An embodiment relates to a setof target genes for RNAi-mediated silencing selected from aninvertebrate genome and where K_(s)>>K_(a) for the selected genes.

In an embodiment, the single- or low-copy-number target genes are asubset of target genes of a first invertebrate species selected from alarger set of genes from the first invertebrate species, wherein theselection is by a sequence comparison performed by a computer betweenthe larger set of genes from the first invertebrate species and a set ofgenes from a second invertebrate species that have been identified assingle- or low-copy-number in the second invertebrate species. In aspecific embodiment, the single- or low-copy-number target genes are asubset of Leptinotarsa decemlineata target genes selected from a largerset of Leptinotarsa decemlineata target genes, wherein the selection isby a sequence comparison performed by a computer between the larger setof Leptinotarsa decemlineata target genes and a set of genes from asecond invertebrate species that have been identified as single- orlow-copy-number in the second invertebrate species. The Leptinotarsadecemlineata single- or low-copy-number target genes selected by themethod are particularly useful in making polynucleotides of thisinvention, including recombinant DNA constructs useful, e.g., forproviding plants having increased resistance to a Leptinotarsa speciesinfestation, and isolated recombinant RNA molecules useful, e.g., inmaking compositions for topical treatment of a plant or Leptinotarsaspecies to provide prevention or control of a Leptinotarsa speciesinfestations. In an embodiment, Leptinotarsa decemlineata single- orlow-copy-number target genes selected by the method are genes having asequence selected from the group consisting of SEQ ID NOs:1-725.

A further aspect of this invention are polyclonal or monoclonalantibodies that bind a protein encoded by a sequence or a fragment of asequence selected from the group consisting of the Target Gene SequencesGroup and polyclonal or monoclonal antibodies that bind a proteinencoded by a sequence or a fragment of a sequence selected from theTrigger Sequences Group, or the complement thereof; such antibodies aremade by routine methods as known to one of ordinary skill in the art,for example using routine protocols as described in “Antibody Methodsand Protocols” (Proetzel and Ebersbach, editors, 2012, Humana Press, NewYork) or “Making and Using Antibodies” (Howard and Kaser, editors, 2006,CRC Press, Boca Raton).

Selection of Effective Polynucleotides by “Tiling”

Polynucleotides of use in the embodiments described herein need not beof the full length of a target gene, and in many embodiments are muchshorter than the target gene. An example of a technique that is usefulfor selecting effective polynucleotides is “tiling”, or evaluation ofpolynucleotides corresponding to adjacent or partially overlappingsegments of a target gene.

In some embodiments, effective polynucleotide triggers can be identifiedby “tiling” gene targets in selected length fragments, e.g., fragmentsof 200-300 nucleotides in length, with partially overlapping regions,e.g., of about 25 nucleotides, along the length of the target gene. Insome embodiments, polynucleotide trigger sequences are designed tocorrespond to (have a nucleotide identity or complementarity with)regions that are unique to the target gene. In some embodiments, theselected region of the target gene can include coding sequence ornon-coding sequence (e.g., promoter regions, 3′ untranslated regions,introns and the like) or a combination of both.

Where it is of interest to design a target effective in suppressingmultiple target genes, the multiple target gene sequences are alignedand polynucleotide triggers are designed to correspond to regions withhigh sequence homology in common among the multiple targets. Conversely,where it is of interest to design a target effective in selectivelysuppressing one among multiple target sequences, the multiple targetgene sequences are aligned and polynucleotide triggers designed tocorrespond to regions with no or low sequence homology in common amongthe multiple targets.

Thermodynamic Considerations in Selection of Effective Polynucleotides

In some embodiments, polynucleotide triggers can be designed or theirsequence optimised using thermodynamic considerations. For example,polynucleotide triggers can be selected based on the thermodynamicscontrolling hybridization between one nucleic acid strand (e.g., apolynucleotide trigger or an individual siRNA) and another (e.g., atarget gene transcript).

Methods and algorithms to predict nucleotide sequences that are likelyto be effective at RNAi-mediated silencing of a target gene are known inthe art. Non-limiting examples of such methods and algorithms include“i-score”, described by Ichihara et al. (2007) Nucleic Acids Res.,35(18): 123e; “Oligowalk”, publicly available atrna.urmc.rochester.edu/servers/oligowalk and described by Lu et al.(2008) Nucleic Acids Res., 36:W104-108; and “Reynolds score”, describedby Khovorova et al. (2004) Nature Biotechnol., 22:326-330.

Permitted Mismatches

By “essentially identical” or “essentially complementary” is meant thata polynucleotide (or at least one strand of a double-strandedpolynucleotide) has sufficient identity or complementarity to the targetgene or to the RNA transcribed from a target gene (e.g., the transcript)to suppress expression of a target gene (e.g., to effect a reduction inlevels or activity of the target gene transcript and/or encodedprotein). Polynucleotides as described herein need not have 100 percentidentity or complementarity to a target gene or to the RNA transcribedfrom a target gene to suppress expression of the target gene (e.g., toeffect a reduction in levels or activity of the target gene transcriptor encoded protein, or to provide control of a Leptinotarsa species). Insome embodiments, the polynucleotide or a portion thereof is designed tobe essentially identical to, or essentially complementary to, a sequenceof at least 18 or 19 contiguous nucleotides in either the target gene orthe RNA transcribed from the target gene. In some embodiments, thepolynucleotide or a portion thereof is designed to be 100% identical to,or 100% complementary to, one or more sequences of 21 contiguousnucleotides in either the target gene or the RNA transcribed from thetarget gene. In certain embodiments, an “essentially identical”polynucleotide has 100 percent sequence identity or at least about 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent sequence identity when compared to the sequence of 18 or morecontiguous nucleotides in either the endogenous target gene or to an RNAtranscribed from the target gene. In certain embodiments, an“essentially complementary” polynucleotide has 100 percent sequencecomplementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity whencompared to the sequence of 18 or more contiguous nucleotides in eitherthe target gene or RNA transcribed from the target gene.

Polynucleotides containing mismatches to the target gene or transcriptcan be used in certain embodiments of the compositions and methodsdescribed herein. In some embodiments, the polynucleotide includes atleast 18 or at least 19 or at least 21 contiguous nucleotides that areessentially identical or essentially complementary to a segment ofequivalent length in the target gene or target gene's transcript. Incertain embodiments, a polynucleotide of 21 or more contiguousnucleotides that is essentially identical or essentially complementaryto a segment of equivalent length in the target gene or target gene'stranscript can have 1 or 2 mismatches to the target gene or transcript(i.e., 1 or 2 mismatches between the polynucleotide's 21 contiguousnucleotides and the segment of equivalent length in the target gene ortarget gene's transcript). In certain embodiments, a polynucleotide ofabout 50, 100, 150, 200, 250, 300, 350 or more nucleotides that containsa contiguous 21 nucleotide span of identity or complementarity to asegment of equivalent length in the target gene or target gene'stranscript can have 1 or 2 or more mismatches to the target gene ortranscript.

In designing polynucleotides with mismatches to an endogenous targetgene or to an RNA transcribed from the target gene, mismatches ofcertain types and at certain positions that are more likely to betolerated can be used. In certain embodiments, mismatches formed betweenadenine and cytosine or guanosine and uracil residues are used asdescribed by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. In someembodiments, mismatches in 19 base-pair overlap regions are located atthe low tolerance positions 5, 7, 8 or 11 (from the 5′ end of a19-nucleotide target), at medium tolerance positions 3, 4, and 12-17(from the 5′ end of a 19-nucleotide target), and/or at the hightolerance positions at either end of the region of complementarity,i.e., positions 1, 2, 18, and 19 (from the 5′ end of a 19-nucleotidetarget) as described by Du et al. (2005) Nucleic Acids Res.,33:1671-1677. Tolerated mismatches can be empirically determined inroutine assays, e.g., in in vitro dietary assays on Leptinotarsa specieslarvae.

Embedding Silencing Elements in Neutral Sequence

In some embodiments, a silencing element comprising a sequencecorresponding to the target gene and which is responsible for anobserved suppression of the target gene is embedded in “neutral”sequence, i.e., inserted into additional nucleotides that have nosequence identity or complementarity to the target gene. Neutralsequence can be desirable, e.g., to increase the overall length of apolynucleotide. For example, it can be desirable for a polynucleotide tobe of a particular size for reasons of stability, cost-effectiveness inmanufacturing, or biological activity. In some embodiments, neutralsequence is also useful in forming the loop in a hairpin trigger or as aspacer between trigger regions.

It has been reported that in another coleopteran species, Diabroticavirgifera, dsRNAs greater than or equal to approximately 60 base-pairs(bp) are required for biological activity in artificial diet bioassays;see Bolognesi et al. (2012) PLoS ONE 7(10): e47534.doi:10.1371/journal.pone.0047534. Thus, in one embodiment, a21-base-pair dsRNA silencing element corresponding to a target gene inTable 1 and found to provide control of a Leptinotarsa infestation isembedded in neutral sequence of an additional 39 base pairs, thusforming a polynucleotide of about 60 base pairs. In some embodiments,the dsRNA trigger includes neutral sequence of between about 60 to about500, or between 100 to about 450 base-pairs, in which is embedded atleast one segment of 21 contiguous nucleotides with a sequence of 100%identity or 100% complementarity with a fragment of equivalent length ofa target gene having a sequence selected from the group consisting ofSEQ ID NOs:1-725 and SEQ ID NOs:726-830 and SEQ ID NOs:1087-1094. Inanother embodiment, a single 21-base-pair silencing element with asequence of 100% identity or 100% complementarity with a fragment ofequivalent length of a target gene is found to be efficacious whenembedded in larger sections of neutral sequence, e.g., where the totalpolynucleotide length is from about 60 to about 300 base pairs. Inanother embodiment, at least one segment of at least 21 contiguousnucleotides of a sequence selected from the group consisting of: SEQ IDNOs:831-1085, 1095-1104, and 1110-1114, or the complement thereof, isembedded in larger sections of neutral sequence to provide anefficacious polynucleotide. In another embodiment, segments frommultiple sequences (or multiple copies of a segment from one or moresequences) selected from the group consisting of: SEQ ID NOs:831-1085,1095-1104, and 1110-1114, or the complement thereof, are embedded inlarger sections of neutral sequence to provide an efficaciouspolynucleotide. In embodiments where the polynucleotide includes regionsof neutral sequence, the polynucleotide will have relatively low overallsequence identity in comparison to the target gene; for example, a dsRNAwith an overall length of 210 base-pairs, containing a single21-base-pair trigger (of 100% identity or complementarity to a21-nucleotide fragment of a target gene) embedded in an additional 189base-pairs of neutral sequence, will have an overall sequence identitywith the target gene of about 10%.

Insecticidal Double-Stranded RNA Molecules

Another aspect of this invention provides an insecticidaldouble-stranded RNA molecule that causes mortality or stunting of growthin a Leptinotarsa species when ingested or contacted by the Leptinotarsaspecies, wherein the insecticidal double-stranded RNA molecule comprisesat least one segment of 18 or more contiguous nucleotides that isessentially identical or essentially complementary to a segment ofequivalent length of a target gene or DNA (cDNA) having a sequenceselected from The Target Gene Sequences Group. In some embodiments, theinsecticidal double-stranded RNA molecule is between about 50 to about500 base-pairs in length. In some embodiments, the insecticidaldouble-stranded RNA molecule comprises at least one segment of at least30 contiguous nucleotides in length. In some embodiments, theinsecticidal double-stranded RNA molecule comprises multiple segments of18 or more contiguous nucleotides that are essentially identical oressentially complementary to a segment of equivalent length of a targetgene or DNA (cDNA) having a sequence selected from The Target GeneSequences Group, wherein the segments are from different regions of thetarget gene (e.g., the segments can correspond to different exon regionsof the target gene, and “spacer” nucleotides which do not correspond toa target gene can optionally be used in between or adjacent to thesegments), or are from different target genes. In some embodiments, theinsecticidal double-stranded RNA molecule comprises multiple segments of18 or more contiguous nucleotides that are essentially identical oressentially complementary to a segment of equivalent length of a targetgene or DNA (cDNA) having a sequence selected from The Target GeneSequences Group, wherein the segments are from different regions of thetarget gene and are arranged in the insecticidal double-stranded RNAmolecule in an order different from the order in which the segmentsnaturally occur in the target gene. In some embodiments, theinsecticidal double-stranded RNA molecule comprises multiple segmentseach of 21 contiguous nucleotides with a sequence of 100% identity or100% complementary to a segment of equivalent length of a target gene orDNA (cDNA) having a sequence selected from The Target Gene SequencesGroup, wherein the segments are from different regions of the targetgene and are arranged in the insecticidal double-stranded RNA moleculein an order different from the order in which the segments naturallyoccur in the target gene. In some embodiments, the insecticidaldouble-stranded RNA molecule comprises one strand comprising a sequenceselected from the group consisting of: SEQ ID NOs:831-1085, 1095-1104,and 1110-1114, or the complement thereof, or comprises an RNA hairpinencoded by a sequence selected from the group consisting of SEQ IDNOs:1105-1109. In some embodiments the insecticidal double-stranded RNAcomprises a dsRNA with a strand having a sequence selected from thegroup consisting of the Trigger Sequences Group. The insecticidaldouble-stranded RNA molecule can be topically applied to a plant,especially a solanaceous plant such as tomato, eggplant, or potato, tocontrol or prevent infestation by a Leptinotarsa species. Theinsecticidal double-stranded RNA molecule can be provided in a formsuitable for ingestion or direct contact by a Leptinotarsa species,e.g., in the form of a spray or powder or bait. Other methods andsuitable compositions for providing the insecticidal double-stranded RNAmolecule are similar to those described in the preceding paragraphs forother aspects of this invention.

Several embodiments relate to a tank mixture comprising one or moreinsecticidal polynucleotides and water or other solvent, optionallyincluding a cationic lipid or an organosilicone surfactant or both.Embodiments include tank mixture formulations of the polynucleotide andoptionally at least one pesticidal agent selected from the groupconsisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein.Embodiments of such compositions include those where one or moreinsecticidal polynucleotides are provided in a living or deadmicroorganism such as a bacterium or fungal or yeast cell, or providedas a microbial fermentation product, or provided in a living or deadplant cell, or provided as a synthetic recombinant polynucleotide. In anembodiment the composition includes a non-pathogenic strain of amicroorganism that contains a polynucleotide as described herein;ingestion or intake of the microorganism results in stunting ormortality of the Leptinotarsa species; non-limiting examples of suitablemicroorganisms include E. coli, B. thuringiensis, Pseudomonas sp.,Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and relatedSerratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae,Clostridium bifermentans and other Clostridium species, or otherspore-forming gram-positive bacteria. In an embodiment, the compositionincludes a plant virus vector comprising a polynucleotide as describedherein; feeding by a Leptinotarsa species on a plant treated with theplant virus vector results in stunting or mortality of the Leptinotarsaspecies. In an embodiment, the composition includes a baculovirus vectorincluding a polynucleotide as described herein; ingestion or intake ofthe vector results in stunting or mortality of the Leptinotarsa species.In an embodiment, a polynucleotide as described herein is encapsulatedin a synthetic matrix such as a polymer or attached to particulates andtopically applied to the surface of a plant; feeding by a Leptinotarsaspecies on the topically treated plant results in stunting or mortalityof the Leptinotarsa species. In an embodiment, a polynucleotide asdescribed herein is provided in the form of a plant cell (e.g., atransgenic solanaceous plant cell of this invention) expressing thepolynucleotide; ingestion of the plant cell or contents of the plantcell by a Leptinotarsa species results in stunting or mortality of theLeptinotarsa species.

In some embodiments, one or more polynucleotides as described herein areprovided with appropriate stickers and wetters required for efficientfoliar coverage as well as UV protectants to protect polynucleotidessuch as dsRNAs from UV damage. Such additives are commonly used in thebioinsecticide industry and are known to those skilled in the art.Compositions for soil application can include granular formulations thatserve as bait for Leptinotarsa species larvae. In some embodiments, oneor more polynucleotides as described herein are further provided with acarrier agent, a surfactant, a cationic lipid (such as that disclosed inExample 18 of U.S. patent application publication 2011/0296556,incorporated by reference herein), an organosilicone, an organosiliconesurfactant, a polynucleotide herbicidal molecule, a non-polynucleotideherbicidal molecule, a non-polynucleotide pesticide, a safener, and aninsect growth regulator. In some embodiments, the composition furtherincludes at least one pesticidal agent selected from the groupconsisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein.

Such compositions are applied in any convenient manner, e.g., byspraying or dusting the Leptinotarsa species directly, or spraying ordusting a plant or environment wherein prevention or control ofinfestation by that Leptinotarsa species is desired, or by applying acoating to a surface of a plant, or by applying a coating to a seed (orseed potato) in preparation for the seed's planting, or by applying asoil drench around roots of a plant for which prevention or control ofinfestation by that Leptinotarsa species is desired.

An effective amount of a polynucleotide as described herein is an amountsufficient to provide control of the Leptinotarsa species, or to preventinfestation by the Leptinotarsa species; determination of effectiveamounts of a polynucleotide are made using routine assays such as thosedescribed in Examples 5 and 6. While there is no upper limit on theconcentrations and dosages of an insecticidal polynucleotide that can beuseful in the methods and compositions provided herein, lower effectiveconcentrations and dosages will generally be sought for efficiency andeconomy. Non-limiting embodiments of effective amounts of apolynucleotide include a range from about 10 nanograms per milliliter toabout 100 micrograms per milliliter of a polynucleotide in a liquid formsprayed on a plant, or from about 10 milligrams per acre to about 100grams per acre of polynucleotide applied to a field of plants, or fromabout 0.001 to about 0.1 microgram per milliliter of polynucleotide inan artificial diet for feeding the Leptinotarsa species. Wherepolynucleotides as described herein are topically applied to a plant,the concentrations can be adjusted in consideration of the volume ofspray or treatment applied to plant leaves or other plant part surfaces,such as flower petals, stems, tubers, fruit, anthers, pollen, leaves,roots, or seeds. In one embodiment, a useful treatment for herbaceousplants using 25-mer polynucleotides as described herein is about 1nanomole (nmol) of polynucleotides per plant, for example, from about0.05 to 1 nmol polynucleotides per plant. Other embodiments forherbaceous plants include useful ranges of about 0.05 to about 100 nmol,or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol ofpolynucleotides per plant. In certain embodiments, about 40 to about 50nmol of a ssDNA polynucleotide are applied. In certain embodiments,about 0.5 nmol to about 2 nmol of a dsRNA is applied. In certainembodiments, a composition containing about 0.5 to about 2.0 milligramsper milliliter, or about 0.14 milligrams per milliliter of a dsRNA or anssDNA (21-mer) is applied. In certain embodiments, a composition ofabout 0.5 to about 1.5 milligrams per milliliter of a dsRNApolynucleotide of this invention of about 50 to about 200 or morenucleotides is applied. In certain embodiments, about 1 nmol to about 5nmol of a dsRNA of this invention is applied to a plant. In certainembodiments, the polynucleotide composition as topically applied to theplant contains at least one polynucleotide of this invention at aconcentration of about 0.01 to about 10 milligrams per milliliter, orabout 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2milligrams per milliliter. Very large plants, trees, or vines canrequire correspondingly larger amounts of polynucleotides. When usinglong dsRNA molecules of this invention that can be processed intomultiple oligonucleotides (e.g., multiple triggers encoded by a singlerecombinant DNA molecule of this invention), lower concentrations can beused. Non-limiting examples of effective polynucleotide treatmentregimes include a treatment of between about 0.1 to about 1 nmol ofpolynucleotide molecule per plant, or between about 1 nmol to about 10nmol of polynucleotide molecule per plant, or between about 10 nmol toabout 100 nmol of polynucleotide molecule per plant.

In some embodiments, one or more polynucleotides is provided with a“transfer agent”, which is an agent that enables a topically appliedpolynucleotide to enter the cells of an organism. Such transfer agentscan be incorporated as part of a composition comprising a polynucleotideas described herein, or can be applied prior to, contemporaneously with,or following application of the polynucleotide. In some embodiments, atransfer agent is an agent that improves the uptake of a polynucleotideof this invention by a Leptinotarsa species. In some embodiments, atransfer agent is an agent that conditions the surface of plant tissue,e.g., seeds, leaves, stems, roots, flowers, or fruits, to permeation bya polynucleotide into plant cells. In some embodiments, the transferagent enables a pathway for a polynucleotide through cuticle waxbarriers, stomata, and/or cell wall or membrane barriers into plantcells.

Suitable transfer agents include agents that increase permeability ofthe exterior of the organism or that increase permeability of cells ofthe organism to polynucleotides. Suitable transfer agents include achemical agent, or a physical agent, or combinations thereof. Chemicalagents for conditioning or transfer include (a) surfactants, (b) anorganic solvent or an aqueous solution or aqueous mixtures of organicsolvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g)enzymes, or any combination thereof. In some embodiments, application ofa polynucleotide and a transfer agent optionally includes an incubationstep, a neutralization step (e.g., to neutralize an acid, base, oroxidizing agent, or to inactivate an enzyme), a rinsing step, orcombinations thereof. Suitable transfer agents can be in the form of anemulsion, a reverse emulsion, a liposome, or other micellar-likecomposition, or can cause the polynucleotide to take the form of anemulsion, a reverse emulsion, a liposome, or other micellar-likecomposition. Embodiments of transfer agents include counter-ions orother molecules that are known to associate with nucleic acid molecules,e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions,polyamines such as spermine, spermidine, or putrescine, and othercations. Embodiments of transfer agents include organic solvents such asDMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide,acetonitrile, dioxane, polypropylene glycol, or other solvents misciblewith water or that dissolve phosphonucleotides in non-aqueous systems(such as is used in synthetic reactions). Embodiments of transfer agentsinclude naturally derived or synthetic oils with or without surfactantsor emulsifiers, e.g., plant-sourced oils, crop oils (such as thoselisted in the 9^(th) Compendium of Herbicide Adjuvants, publiclyavailable on-line at herbicide.adjuvants.com), paraffinic oils, polyolfatty acid esters, or oils with short-chain molecules modified withamides or polyamines such as polyethyleneimine or N-pyrrolidine.

Embodiments of transfer agents include organosilicone preparations. Forexample, a suitable transfer agent is an organosilicone preparation thatis commercially available as SILWET L-77® brand surfactant having CASNumber 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, andcurrently available from Momentive Performance Materials, Albany, N.Y.One embodiment includes a composition that comprises a polynucleotideand a transfer agent including an organosilicone preparation such asSilwet L-77 in the range of about 0.015 to about 2 percent by weight (wtpercent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent). Oneembodiment includes a composition that comprises a polynucleotide ofthis invention and a transfer agent including SILWET L-77® brandsurfactant in the range of about 0.3 to about 1 percent by weight (wtpercent) or about 0.5 to about 1%, by weight (wt percent).

Organosilicone compounds useful as transfer agents for use in thisinvention include, but are not limited to, compounds that include: (a) atrisiloxane head group that is covalently linked to, (b) an alkyl linkerincluding, but not limited to, an n-propyl linker, that is covalentlylinked to, (c) a polyglycol chain, that is covalently linked to, (d) aterminal group. Trisiloxane head groups of such organosilicone compoundsinclude, but are not limited to, heptamethyltrisiloxane. Alkyl linkerscan include, but are not limited to, an n-propyl linker. Polyglycolchains include, but are not limited to, polyethylene glycol orpolypropylene glycol. Polyglycol chains can comprise a mixture thatprovides an average chain length “n” of about “7.5”. In certainembodiments, the average chain length “n” can vary from about 5 to about14. Terminal groups can include, but are not limited to, alkyl groupssuch as a methyl group. Organosilicone compounds useful as transferagents include, but are not limited to, trisiloxane ethoxylatesurfactants or polyalkylene oxide modified heptamethyl trisiloxane. Anexample of a transfer agent for use in this invention is Compound I:

Organosilicone compounds useful as transfer agents are used, e.g., asfreshly made concentrations in the range of about 0.015 to about 2percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025,0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wtpercent).

Embodiments of transfer agents include one or more salts such asammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate,provided in or used with a composition including a polynucleotide. Insome embodiments, ammonium chloride, tetrabutylphosphonium bromide,and/or ammonium sulfate are used at a concentration of about 0.5% toabout 5% (w/v), or about 1% to about 3% (w/v), or about 2% (w/v). Incertain embodiments, the composition including a polynucleotide includesan ammonium salt at a concentration greater or equal to 300 millimolar.In certain embodiments, the composition including a polynucleotideincludes an organosilicone transfer agent in a concentration of about0.015 to about 2 percent by weight (wt percent) as well as ammoniumsulfate at concentrations from about 80 to about 1200 mM or about 150 mMto about 600 mM.

Embodiments of transfer agents include a phosphate salt. Phosphate saltsuseful in a composition including a polynucleotide include, but are notlimited to, calcium, magnesium, potassium, or sodium phosphate salts. Incertain embodiments, a composition including a polynucleotide includes aphosphate salt at a concentration of at least about 5 millimolar, atleast about 10 millimolar, or at least about 20 millimolar. In certainembodiments, a composition including a polynucleotide a phosphate saltin a range of about 1 mM to about 25 mM or in a range of about 5 mM toabout 25 mM. In certain embodiments, the composition including apolynucleotide sodium phosphate at a concentration of at least about 5millimolar, at least about 10 millimolar, or at least about 20millimolar. In certain embodiments, a composition including apolynucleotide includes sodium phosphate at a concentration of about 5millimolar, about 10 millimolar, or about 20 millimolar. In certainembodiments, a composition including a polynucleotide includes a sodiumphosphate salt in a range of about 1 mM to about 25 mM or in a range ofabout 5 mM to about 25 mM. In certain embodiments, a compositionincluding a polynucleotide includes a sodium phosphate salt in a rangeof about 10 mM to about 160 mM or in a range of about 20 mM to about 40mM. In certain embodiments, a composition including a polynucleotideincludes a sodium phosphate buffer at a pH of about 6.8.

Embodiments of transfer agents include surfactants and/or effectivemolecules contained therein. Surfactants and/or effective moleculescontained therein include, but are not limited to, sodium or lithiumsalts of fatty acids (such as tallow or tallowamines or phospholipids)and organosilicone surfactants. In certain embodiments, a compositionincluding a polynucleotide is formulated with counter-ions or othermolecules that are known to associate with nucleic acid molecules.Non-limiting examples include, tetraalkyl ammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such asspermine, spermidine, or putrescine. In certain embodiments, acomposition including a polynucleotide is formulated with anon-polynucleotide herbicide e.g., glyphosate, auxin-like benzoic acidherbicides including dicamba, chloramben, and TBA, glufosinate,auxin-like herbicides including phenoxy carboxylic acid herbicide,pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide,pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide,sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione,protoporphyrinogen oxidase inhibitors, and4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides. In certainembodiments, a composition including a polynucleotide is formulated witha non-polynucleotide pesticide, e.g., a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein. In some embodiments, a composition including apolynucleotide and a non-polynucleotide pesticide provides synergeticimprovement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with thepolynucleotide alone or the non-polynucleotide pesticide alone. In someembodiments, a composition comprising a double-stranded RNA with astrand having a sequence selected from the group consisting of theTrigger Sequences Group is combined with a non-polynucleotide pesticide(e.g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillusthuringiensis insecticidal protein, a Xenorhabdus insecticidal protein,a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, and a Bacillus sphaericus insecticidal protein),wherein the combination is found to effect synergistically improvedprevention or control of Leptinotarsa species infestations, whencompared to the effect obtained with the double-stranded RNA alone orthe non-polynucleotide pesticide alone.

Related Techniques

Embodiments of the polynucleotides and nucleic acid molecules asdescribed herein can include additional elements, such as promoters,small RNA recognition sites, aptamers or ribozymes, additional andadditional expression cassettes for expressing coding sequences (e.g.,to express a transgene such as an insecticidal protein or selectablemarker) or non-coding sequences (e.g., to express additional suppressionelements). For example, an aspect of this invention provides arecombinant DNA construct comprising a heterologous promoter operablylinked to DNA comprising at least one segment of 18 or more contiguousnucleotides with a sequence of about 95% to about 100% identity with afragment of equivalent length of a DNA having a sequence selected fromthe Target Gene Sequences Group or the DNA complement thereof. Anotheraspect of the invention provides a recombinant DNA construct comprisinga heterologous promoter operably linked to DNA encoding an RNA hairpinhaving an anti-sense region having a sequence, or a fragment of asequence, selected from the group selected from the Trigger SequencesGroup. In another embodiment, a recombinant DNA construct comprising apromoter operably linked to DNA encoding: (a) an RNA silencing elementfor suppressing a target gene selected from the group consisting of thegenes identified in Table 1), and (b) an aptamer, is stably integratedinto the plant's genome from where RNA transcripts including the RNAaptamer and the RNA silencing element are expressed in cells of theplant; the aptamer serves to guide the RNA silencing element to adesired location in the cell. In another embodiment, inclusion of one ormore recognition sites for binding and cleavage by a small RNA (e.g., bya miRNA or an siRNA that is expressed only in a particular cell ortissue) allows for more precise expression patterns in a plant, whereinthe expression of the recombinant DNA construct is suppressed where thesmall RNA is expressed. Such additional elements are described below.

Promoters

Promoters of use in the invention are functional in the cell in whichthe construct is intended to be transcribed. Generally these promotersare heterologous promoters, as used in recombinant constructs, i.e.,they are not in nature found to be operably linked to the other nucleicelements used in the constructs described herein. In variousembodiments, the promoter is selected from the group consisting of aconstitutive promoter, a spatially specific promoter, a temporallyspecific promoter, a developmentally specific promoter, and an induciblepromoter. In many embodiments the promoter is a promoter functional in aplant, for example, a pol II promoter, a pol III promoter, a pol IVpromoter, or a pol V promoter.

Non-constitutive promoters suitable for use with the recombinant DNAconstructs of this invention include spatially specific promoters,temporally specific promoters, and inducible promoters. Spatiallyspecific promoters can include organelle-, cell-, tissue-, ororgan-specific promoters (e.g., a plastid-specific, a root-specific, apollen-specific, or a seed-specific promoter for expression in plastids,roots, pollen, or seeds, respectively). In many cases a seed-specific,embryo-specific, aleurone-specific, or endosperm-specific promoter isespecially useful. Temporally specific promoters can include promotersthat tend to promote expression during certain developmental stages in aplant's growth cycle, or during different times of day or night, or atdifferent seasons in a year. Inducible promoters include promotersinduced by chemicals or by environmental conditions such as, but notlimited to, biotic or abiotic stress (e.g., water deficit or drought,heat, cold, high or low nutrient or salt levels, high or low lightlevels, or pest or pathogen infection). MicroRNA promoters are useful,especially those having a temporally specific, spatially specific, orinducible expression pattern; examples of miRNA promoters, as well asmethods for identifying miRNA promoters having specific expressionpatterns, are provided in U.S. Patent Application Publications2006/0200878, 2007/0199095, and 2007/0300329, which are specificallyincorporated herein by reference. An expression-specific promoter canalso include promoters that are generally constitutively expressed butat differing degrees or “strengths” of expression, including promoterscommonly regarded as “strong promoters” or as “weak promoters”.

Promoters of particular interest include the following examples: anopaline synthase promoter isolated from T-DNA of Agrobacterium; acauliflower mosaic virus 35S promoter; enhanced promoter elements orchimeric promoter elements such as an enhanced cauliflower mosaic virus(CaMV) 35S promoter linked to an enhancer element (an intron from heatshock protein 70 of Zea mays); root specific promoters such as thosedisclosed in U.S. Pat. Nos. 5,837,848; 6,437,217 and 6,426,446; a maizeL3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252; a promoter fora plant nuclear gene encoding a plastid-localized aldolase disclosed inU.S. Patent Application Publication 2004/0216189; cold-induciblepromoters disclosed in U.S. Pat. No. 6,084,089; salt-inducible promotersdisclosed in U.S. Pat. No. 6,140,078; light-inducible promotersdisclosed in U.S. Pat. No. 6,294,714; pathogen-inducible promotersdisclosed in U.S. Pat. No. 6,252,138; and water deficit-induciblepromoters disclosed in U.S. Patent Application Publication 2004/0123347A1. All of the above-described patents and patent publicationsdisclosing promoters and their use, especially in recombinant DNAconstructs functional in plants are incorporated herein by reference.

Plant vascular- or phloem-specific promoters of interest include a rolCor rolA promoter of Agrobacterium rhizogenes, a promoter of aAgrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1gene promoter, a Commelina yellow mottle badnavirus promoter, a coconutfoliar decay virus promoter, a rice tungro bacilliform virus promoter,the promoter of a pea glutamine synthase GS3A gene, a invCD111 andinvCD141 promoters of a potato invertase genes, a promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88:5212-5216, aVAHOX1 promoter region, a pea cell wall invertase gene promoter, an acidinvertase gene promoter from carrot, a promoter of a sulfate transportergene Sultr1;3, a promoter of a plant sucrose synthase gene, and apromoter of a plant sucrose transporter gene.

Promoters suitable for use with a recombinant DNA construct orpolynucleotide of this invention include polymerase II (“pol II”)promoters and polymerase III (“pol III”) promoters. RNA polymerase IItranscribes structural or catalytic RNAs that are usually shorter than400 nucleotides in length, and recognizes a simple run of T residues asa termination signal; it has been used to transcribe siRNA duplexes(see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Pol IIpromoters are therefore in certain embodiments where a short RNAtranscript is to be produced from a recombinant DNA construct of thisinvention. In one embodiment, the recombinant DNA construct comprises apol II promoter to express an RNA transcript flanked by self-cleavingribozyme sequences (e.g., self-cleaving hammerhead ribozymes), resultingin a processed RNA, such as a single-stranded RNA that binds to thetranscript of the Leptinotarsa target gene, with defined 5′ and 3′ ends,free of potentially interfering flanking sequences. An alternativeapproach uses pol III promoters to generate transcripts with relativelydefined 5′ and 3′ ends, i.e., to transcribe an RNA with minimal 5′ and3′ flanking sequences. In some embodiments, Pol III promoters (e.g., U6or H1 promoters) are for adding a short AT-rich transcriptiontermination site that results in 2 base-pair overhangs (UU) in thetranscribed RNA; this is useful, e.g., for expression of siRNA-typeconstructs. Use of pol III promoters for driving expression of siRNAconstructs has been reported; see van de Wetering et al. (2003) EMBORep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448.Baculovirus promoters such as baculovirus polyhedrin and p10 promotersare known in the art and commercially available; see, e.g., Invitrogen's“Guide to Baculovirus Expression Vector Systems (BEVS) and Insect CellCulture Techniques”, 2002 (Life Technologies, Carlsbad, Calif.) and F.J. Haines et al. “Baculovirus Expression Vectors”, undated (OxfordExpression Technologies, Oxford, UK).

The promoter element can include nucleic acid sequences that are notnaturally occurring promoters or promoter elements or homologues thereofbut that can regulate expression of a gene. Examples of such “geneindependent” regulatory sequences include naturally occurring orartificially designed RNA sequences that include a ligand-binding regionor aptamer (see “Aptamers”, below) and a regulatory region (which can becis-acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol.,22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343,Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463,Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler etal. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647,and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such“riboregulators” could be selected or designed for specific spatial ortemporal specificity, for example, to regulate translation of DNA thatencodes a silencing element for suppressing a Leptinotarsa target geneonly in the presence (or absence) of a given concentration of theappropriate ligand. One example is a riboregulator that is responsive toan endogenous ligand (e.g., jasmonic acid or salicylic acid) produced bythe plant when under stress (e.g., abiotic stress such as water,temperature, or nutrient stress, or biotic stress such as attach bypests or pathogens); under stress, the level of endogenous ligandincreases to a level sufficient for the riboregulator to begintranscription of the DNA that encodes a silencing element forsuppressing a Leptinotarsa target gene.

Recombinase Sites

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA encoding one or more site-specificrecombinase recognition sites. In one embodiment, the recombinant DNAconstruct comprises at least a pair of loxP sites, wherein site-specificrecombination of DNA between the loxP sites is mediated by a Crerecombinase. The position and relative orientation of the loxP sites isselected to achieve the desired recombination; for example, when theloxP sites are in the same orientation, the DNA between the loxP sitesis excised in circular form. In another embodiment, the recombinant DNAconstruct comprises DNA encoding one loxP site; in the presence of Crerecombinase and another DNA with a loxP site, the two DNAs arerecombined.

Aptamers

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA that is processed to an RNA aptamer, thatis, an RNA that binds to a ligand through binding mechanism that is notprimarily based on Watson-Crick base-pairing (in contrast, for example,to the base-pairing that occurs between complementary, anti-parallelnucleic acid strands to form a double-stranded nucleic acid structure).See, for example, Ellington and Szostak (1990) Nature, 346:818-822.Examples of aptamers can be found, for example, in the public AptamerDatabase, available on line at aptamer.icmb.utexas.edu (Lee et al.(2004) Nucleic Acids Res., 32(1):D95-100). Aptamers useful in theinvention can, however, be monovalent (binding a single ligand) ormultivalent (binding more than one individual ligand, e.g., binding oneunit of two or more different ligands).

Ligands useful in the invention include any molecule (or part of amolecule) that can be recognized and be bound by a nucleic acidsecondary structure by a mechanism not primarily based on Watson-Crickbase pairing. In this way, the recognition and binding of ligand andaptamer is analogous to that of antigen and antibody, or of biologicaleffector and receptor. Ligands can include single molecules (or part ofa molecule), or a combination of two or more molecules (or parts of amolecule), and can include one or more macromolecular complexes (e.g.,polymers, lipid bilayers, liposomes, cellular membranes or othercellular structures, or cell surfaces). Examples of specific ligandsinclude vitamins such as coenzyme B₁₂ and thiamine pyrophosphate, flavinmononucleotide, guanine, adenosine, S-adenosylmethionine,S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine,glucosamine-6-phosphate, caffeine, theophylline, antibiotics such aschloramphenicol and neomycin, herbicides such as glyphosate and dicamba,proteins including viral or phage coat proteins and invertebrateepidermal or digestive tract surface proteins, and RNAs including viralRNA, transfer-RNAs (t-RNAs), ribosomal RNA (rRNA), and RNA polymerasessuch as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamersuseful in the invention are “thermoswitches” that do not bind a ligandbut are thermally responsive, that is to say, the aptamer's conformationis determined by temperature; see, for example, Box 3 in Mandal andBreaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

Transgene Transcription Units

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises a transgene transcription unit. A transgenetranscription unit comprises DNA sequence encoding a gene of interest,e.g., a natural protein or a heterologous protein. A gene of interestcan be any coding or non-coding sequence from any species (including,but not limited to, non-eukaryotes such as bacteria, and viruses; fungi,protists, plants, invertebrates, and vertebrates. Particular genes ofinterest are genes encoding at least one pesticidal agent selected fromthe group consisting of a patatin, a plant lectin, a phytoecdysteroid, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein. The transgene transcription unit can furtherinclude 5′ or 3′ sequence or both as required for transcription of thetransgene.

Introns

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA encoding a spliceable intron. By “intron”is generally meant a segment of DNA (or the RNA transcribed from such asegment) that is located between exons (protein-encoding segments of theDNA or corresponding transcribed RNA), wherein, during maturation of themessenger RNA, the intron present is enzymatically “spliced out” orremoved from the RNA strand by a cleavage/ligation process that occursin the nucleus in eukaryotes. The term “intron” is also applied tonon-coding DNA sequences that are transcribed to RNA segments that canbe spliced out of a maturing RNA transcript, but are not introns foundbetween protein-coding exons. One example of these are spliceablesequences that that have the ability to enhance expression in plants (insome cases, especially in monocots) of a downstream coding sequence;these spliceable sequences are naturally located in the 5′ untranslatedregion of some plant genes, as well as in some viral genes (e.g., thetobacco mosaic virus 5′ leader sequence or “omega” leader described asenhancing expression in plant genes by Gallie and Walbot (1992) NucleicAcids Res., 20:4631-4638). These spliceable sequences or“expression-enhancing introns” can be artificially inserted in the 5′untranslated region of a plant gene between the promoter but before anyprotein-coding exons. Examples of such expression-enhancing intronsinclude, but are not limited to, a maize alcohol dehydrogenase(Zm-Adh1), a maize Bronze-1 expression-enhancing intron, a rice actin 1(Os-Act1) intron, a Shrunken-1 (Sh-1) intron, a maize sucrose synthaseintron, a heat shock protein 18 (hsp18) intron, and an 82 kilodaltonheat shock protein (hsp82) intron. U.S. Pat. Nos. 5,593,874 and5,859,347, specifically incorporated by reference herein, describemethods of improving recombinant DNA constructs for use in plants byinclusion of an expression-enhancing intron derived from the 70kilodalton maize heat shock protein (hsp70) in the non-translated leaderpositioned 3′ from the gene promoter and 5′ from the firstprotein-coding exon.

Ribozymes

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA encoding one or more ribozymes. Ribozymesof particular interest include a self-cleaving ribozyme, a hammerheadribozyme, or a hairpin ribozyme. In one embodiment, the recombinant DNAconstruct comprises DNA encoding one or more ribozymes that serve tocleave the transcribed RNA to provide defined segments of RNA, such assilencing elements for suppressing a Leptinotarsa target gene.

Gene Suppression Elements

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA encoding additional gene suppressionelement for suppressing a target gene other than a Leptinotarsa targetgene. The target gene to be suppressed can include coding or non-codingsequence or both.

Suitable gene suppression elements are described in detail in U.S.Patent Application Publication 2006/0200878, which disclosure isspecifically incorporated herein by reference, and include one or moreof:

-   -   (a) DNA that comprises at least one anti-sense DNA segment that        is anti-sense to at least one segment of the gene to be        suppressed;    -   (b) DNA that comprises multiple copies of at least one        anti-sense DNA segment that is anti-sense to at least one        segment of the gene to be suppressed;    -   (c) DNA that comprises at least one sense DNA segment that is at        least one segment of the gene to be suppressed;    -   (d) DNA that comprises multiple copies of at least one sense DNA        segment that is at least one segment of the gene to be        suppressed;    -   (e) DNA that transcribes to RNA for suppressing the gene to be        suppressed by forming double-stranded RNA and comprises at least        one anti-sense DNA segment that is anti-sense to at least one        segment of the gene to be suppressed and at least one sense DNA        segment that is at least one segment of the gene to be        suppressed;    -   (f) DNA that transcribes to RNA for suppressing the gene to be        suppressed by forming a single double-stranded RNA and comprises        multiple serial anti-sense DNA segments that are anti-sense to        at least one segment of the gene to be suppressed and multiple        serial sense DNA segments that are at least one segment of the        gene to be suppressed;    -   (g) DNA that transcribes to RNA for suppressing the gene to be        suppressed by forming multiple double strands of RNA and        comprises multiple anti-sense DNA segments that are anti-sense        to at least one segment of the gene to be suppressed and        multiple sense DNA segments that are at least one segment of the        gene to be suppressed, and wherein the multiple anti-sense DNA        segments and the multiple sense DNA segments are arranged in a        series of inverted repeats;    -   (h) DNA that comprises nucleotides derived from a plant miRNA;    -   (i) DNA that comprises nucleotides of a siRNA;    -   (j) DNA that transcribes to an RNA aptamer capable of binding to        a ligand; and    -   (k) DNA that transcribes to an RNA aptamer capable of binding to        a ligand, and DNA that transcribes to regulatory RNA capable of        regulating expression of the gene to be suppressed, wherein the        regulation is dependent on the conformation of the regulatory        RNA, and the conformation of the regulatory RNA is        allosterically affected by the binding state of the RNA aptamer.

In some embodiments, an intron is used to deliver a gene suppressionelement in the absence of any protein-coding exons (coding sequence). Inone example, an intron, such as an expression-enhancing intron, isinterrupted by embedding within the intron a gene suppression element,wherein, upon transcription, the gene suppression element is excisedfrom the intron. Thus, protein-coding exons are not required to providethe gene suppressing function of the recombinant DNA constructsdisclosed herein.

Transcription Regulatory Elements

In some embodiments, the recombinant DNA construct or polynucleotide ofthis invention comprises DNA encoding a transcription regulatoryelement. Transcription regulatory elements include elements thatregulate the expression level of the recombinant DNA construct of thisinvention (relative to its expression in the absence of such regulatoryelements). Examples of suitable transcription regulatory elementsinclude riboswitches (cis- or trans-acting), transcript stabilizingsequences, and miRNA recognition sites, as described in detail in U.S.Patent Application Publication 2006/0200878, specifically incorporatedherein by reference.

Making and Using Transgenic Plant Cells and Transgenic Plants

Transformation of a plant can include any of several well-known methodsand compositions. Suitable methods for plant transformation includevirtually any method by which DNA can be introduced into a cell. Onemethod of plant transformation is microprojectile bombardment, forexample, as illustrated in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat.No. 5,538,880 (maize), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No.5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No.6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No.6,365,807 (rice), and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No.6,403,865 (maize), all of which are incorporated by reference forenabling the production of transgenic plants.

Another useful method of plant transformation is Agrobacterium-mediatedtransformation by means of Agrobacterium containing a binary Ti plasmidsystem, wherein the Agrobacterium carries a first Ti plasmid and asecond, chimeric plasmid containing at least one T-DNA border of awild-type Ti plasmid, a promoter functional in the transformed plantcell and operably linked to a polynucleotide or recombinant DNAconstruct of this invention. See, for example, the binary systemdescribed in U.S. Pat. No. 5,159,135, incorporated by reference. Alsosee De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al.,(1983) Nature, 303:179. In such a binary system, the smaller plasmid,containing the T-DNA border or borders, can be conveniently constructedand manipulated in a suitable alternative host, such as E. coli, andthen transferred into Agrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include procedures disclosed in U.S. Pat. Nos.5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011,5,569,834, 5,824,877 and 6,384,301 (soybean); U.S. Pat. Nos. 5,591,616and 5,981,840 (maize); U.S. Pat. No. 5,463,174 (brassicas includingcanola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No. 6,329,571(rice), and in U.S. Patent Application Publications 2004/0244075 (maize)and 2001/0042257 A1 (sugar beet), all of which are specificallyincorporated by reference for enabling the production of transgenicplants. U.S. Patent Application Publication 2011/0296555 discloses inExample 5 the transformation vectors (including the vector sequences)and detailed protocols for transforming maize, soybean, canola, cotton,and sugarcane) and is specifically incorporated by reference forenabling the production of transgenic plants. Similar methods have beenreported for many plant species, both dicots and monocots, including,among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653);asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A.,84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice(Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) PlantCell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667;Becker et al. (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996)Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant CellPhysiol., 47:426-431). See also a description of vectors, transformationmethods, and production of transformed Arabidopsis thaliana plants wheretranscription factors are constitutively expressed by a CaMV35Spromoter, in U.S. Patent Application Publication 2003/0167537 A1,incorporated by reference. Transformation methods specifically usefulfor solanaceous plants are well known in the art. See, for example,publicly described transformation methods for tomato (Sharma et al.(2009), J. Biosci., 34:423-433), eggplant (Arpaia et al. (1997) Theor.Appl. Genet., 95:329-334), potato (Bannerjee et al. (2006) Plant Sci.,170:732-738; Chakravarty et al. (2007) Amer. J. Potato Res., 84:301-311;S. Millam “Agrobacterium-mediated transformation of potato.” Chapter 19(pp. 257-270), “Transgenic Crops of the World: Essential Protocols”, IanS. Curtis (editor), Springer, 2004), and peppers (Li et al. (2003) PlantCell Reports, 21: 785-788). Stably transgenic potato, tomato, andeggplant have been commercially introduced in various regions; see,e.g., K. Redenbaugh et al. “Safety Assessment of Genetically EngineeredFruits and Vegetables: A Case Study of the FLAVR SAVR Tomato”, CRCPress, Boca Raton, 1992, and the extensive publicly availabledocumentation of commercial genetically modified crops in the GM CropDatabase; see: CERA. (2012). GM Crop Database. Center for EnvironmentalRisk Assessment (CERA), ILSI Research Foundation, Washington D.C.,available electronically at www.cera-gmc.org/?action=gm_crop_database.Various methods of transformation of other plant species are well knownin the art, see, for example, the encyclopedic reference, “Compendium ofTransgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C.Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (availableelectronically atmrw.interscience.wiley.com/emrw/9781405181099/hpt/toc), which describestransformation procedures for cereals and forage grasses (rice, maize,wheat, barley, oat, sorghum, pearl millet, finger millet, cool-seasonforage grasses, and bahiagrass), oilseed crops (soybean, oilseedbrassicas, sunflower, peanut, flax, sesame, and safflower), legumegrains and forages (common bean, cowpea, pea, faba bean, lentil, teparybean, Asiatic beans, pigeonpea, vetch, chickpea, lupin, alfalfa, andclovers), temperate fruits and nuts (apple, pear, peach, plums, berrycrops, cherries, grapes, olive, almond, and Persian walnut), tropicaland subtropical fruits and nuts (citrus, grapefruit, banana andplantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit,and persimmon), vegetable crops (tomato, eggplant, peppers, vegetablebrassicas, radish, carrot, cucurbits, alliums, asparagus, and leafyvegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet,stevia, potato, sweet potato, cassava, and cotton), plantation crops,ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree,medicinal plants, ornamentals, and turf grasses), and forest treespecies.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism.Recipient cell targets include, but are not limited to, meristem cells,callus, immature embryos or parts of embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. Any cell from which a fertileplant can be regenerated is contemplated as a useful recipient cell forpractice of this invention. Callus can be initiated from various tissuesources, including, but not limited to, immature embryos or parts ofembryos, seedling apical meristems, microspores, and the like. Thosecells which are capable of proliferating as callus can serve asrecipient cells for genetic transformation. Practical transformationmethods and materials for making transgenic plants of this invention(e.g., various media and recipient target cells, transformation ofimmature embryos, and subsequent regeneration of fertile transgenicplants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and6,232,526 and U.S. Patent Application Publication 2004/0216189, whichare specifically incorporated by reference.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theantibiotics or herbicides to which a plant cell is resistant can be auseful agent for selection. Potentially transformed cells are exposed tothe selective agent. In the population of surviving cells will be thosecells where, generally, the resistance-conferring gene is integrated andexpressed at sufficient levels to permit cell survival. Cells can betested further to confirm stable integration of the recombinant DNA.Commonly used selective marker genes include those conferring resistanceto antibiotics such as kanamycin or paromomycin (nptII), hygromycin B(aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicidessuch as glufosinate (bar or pat) and glyphosate (EPSPS). Examples ofuseful selective marker genes and selection agents are illustrated inU.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all ofwhich are specifically incorporated by reference. Screenable markers orreporters, such as markers that provide an ability to visually identifytransformants can also be employed. Examples of useful screenablemarkers include, for example, a gene expressing a protein that producesa detectable color by acting on a chromogenic substrate (e.g., betaglucuronidase (GUS) (uidA) or luciferase (luc)) or that itself isdetectable, such as green fluorescent protein (GFP) (gfp) or animmunogenic molecule. Those of skill in the art will recognize that manyother useful markers or reporters are available for use.

Detecting or measuring transcription of a recombinant DNA construct in atransgenic plant cell can be achieved by any suitable method, includingprotein detection methods (e.g., western blots, ELISAs, and otherimmunochemical methods), measurements of enzymatic activity, or nucleicacid detection methods (e.g., Southern blots, northern blots, PCR,RT-PCR, fluorescent in situ hybridization).

Other suitable methods for detecting or measuring transcription in aplant cell of a recombinant polynucleotide of this invention targeting aLeptinotarsa species target gene include measurement of any other traitthat is a direct or proxy indication of the level of expression of thetarget gene in the Leptinotarsa species, relative to the level ofexpression observed in the absence of the recombinant polynucleotide,e.g., growth rates, mortality rates, or reproductive or recruitmentrates of the Leptinotarsa species, or measurements of injury (e.g., rootinjury) or yield loss in a plant or field of plants infested by theLeptinotarsa species. In general, suitable methods for detecting ormeasuring transcription in a plant cell of a recombinant polynucleotideof interest include, e.g., gross or microscopic morphological traits,growth rates, yield, reproductive or recruitment rates, resistance topests or pathogens, or resistance to biotic or abiotic stress (e.g.,water deficit stress, salt stress, nutrient stress, heat or coldstress). Such methods can use direct measurements of a phenotypic traitor proxy assays (e.g., in plants, these assays include plant part assayssuch as leaf or root assays to determine tolerance of abiotic stress).Such methods include direct measurements of resistance to aninvertebrate pest or pathogen (e.g., damage to plant tissues) or proxyassays (e.g., plant yield assays, or bioassays such as the Western cornrootworm (Diabrotica virgifera virgifera LeConte) larval bioassaydescribed in International Patent Application Publication WO2005/110068A2 and U.S. Patent Application Publication US 2006/0021087 A1,specifically incorporated by reference, or the soybean cyst nematodebioassay described by Steeves et al. (2006) Funct. Plant Biol.,33:991-999, wherein cysts per plant, cysts per gram root, eggs perplant, eggs per gram root, and eggs per cyst are measured, or theColorado potato beetle (Leptinotarsa decemlineata) bioassay describedherein in the working Examples.

The recombinant DNA constructs of this invention can be stacked withother recombinant DNA for imparting additional traits (e.g., in the caseof transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance, and thelike) for example, by expressing or suppressing other genes. Constructsfor coordinated decrease and increase of gene expression are disclosedin U.S. Patent Application Publication 2004/0126845 A1, specificallyincorporated by reference.

Seeds of fertile transgenic plants can be harvested and used to growprogeny generations, including hybrid generations, of transgenic plantsof this invention that include the recombinant DNA construct in theirgenome. Thus, in addition to direct transformation of a plant with arecombinant DNA construct of this invention, transgenic plants of thisinvention can be prepared by crossing a first plant having therecombinant DNA with a second plant lacking the construct. For example,the recombinant DNA can be introduced into a plant line that is amenableto transformation to produce a transgenic plant, which can be crossedwith a second plant line to introgress the recombinant DNA into theresulting progeny. A transgenic plant of this invention can be crossedwith a plant line having other recombinant DNA that confers one or moreadditional trait(s) (such as, but not limited to, herbicide resistance,pest or disease resistance, environmental stress resistance, modifiednutrient content, and yield improvement) to produce progeny plantshaving recombinant DNA that confers both the desired target sequenceexpression behavior and the additional trait(s).

In such breeding for combining traits the transgenic plant donating theadditional trait can be a male line (pollinator) and the transgenicplant carrying the base traits can be the female line. The progeny ofthis cross segregate such that some of the plant will carry the DNA forboth parental traits and some will carry DNA for one parental trait;such plants can be identified by markers associated with parentalrecombinant DNA Progeny plants carrying DNA for both parental traits canbe crossed back into the female parent line multiple times, e.g.,usually 6 to 8 generations, to produce a homozygous progeny plant withsubstantially the same genotype as one original transgenic parental lineas well as the recombinant DNA of the other transgenic parental line.

Yet another aspect of this invention is a transgenic plant grown fromthe transgenic seed (or in the case of potatoes, a transgenic seedpotato) of this invention. This invention contemplates transgenic plantsgrown directly from transgenic seed containing the recombinant DNA aswell as progeny generations of plants, including inbred or hybrid plantlines, made by crossing a transgenic plant grown directly fromtransgenic seed to a second plant not grown from the same transgenicseed. Crossing can include, for example, the following steps:

-   -   (a) plant seeds of the first parent plant (e.g., non-transgenic        or a transgenic) and a second parent plant that is transgenic        according to the invention;    -   (b) grow the seeds of the first and second parent plants into        plants that bear flowers;    -   (c) pollinate a flower from the first parent with pollen from        the second parent; and    -   (d) harvest seeds produced on the parent plant bearing the        fertilized flower.

It is often desirable to introgress recombinant DNA into elitevarieties, e.g., by backcrossing, to transfer a specific desirable traitfrom one source to an inbred or other plant that lacks that trait. Thiscan be accomplished, for example, by first crossing a superior inbred(“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent),which carries the appropriate gene(s) for the trait in question, forexample, a construct prepared in accordance with the current invention.The progeny of this cross first are selected in the resultant progenyfor the desired trait to be transferred from the non-recurrent parent“B”, and then the selected progeny are mated back to the superiorrecurrent parent “A”. After five or more backcross generations withselection for the desired trait, the progeny can be essentiallyhemizygous for loci controlling the characteristic being transferred,but are like the superior parent for most or almost all other genes. Thelast backcross generation would be selfed to give progeny which are purebreeding for the gene(s) being transferred, e.g., one or moretransformation events.

Through a series of breeding manipulations, a selected DNA construct canbe moved from one line into an entirely different line without the needfor further recombinant manipulation. One can thus produce inbred plantswhich are true breeding for one or more DNA constructs. By crossingdifferent inbred plants, one can produce a large number of differenthybrids with different combinations of DNA constructs. In this way,plants can be produced which have the desirable agronomic propertiesfrequently associated with hybrids (“hybrid vigor”), as well as thedesirable characteristics imparted by one or more DNA constructs.

In certain transgenic plant cells and transgenic plants of thisinvention, it is sometimes desirable to concurrently express a gene ofinterest while also modulating expression of a Leptinotarsa target gene.Thus, in some embodiments, the transgenic plant contains recombinant DNAfurther comprising a gene expression element for expressing at least onegene of interest, and transcription of the recombinant DNA construct ofthis invention is effected with concurrent transcription of the geneexpression element.

In some embodiments, the recombinant DNA constructs of this inventioncan be transcribed in any plant cell or tissue or in a whole plant ofany developmental stage. Transgenic plants can be derived from anymonocot or dicot plant, such as, but not limited to, plants ofcommercial or agricultural interest, such as crop plants (especiallycrop plants used for human food or animal feed), wood- or pulp-producingtrees, vegetable plants, fruit plants, and ornamental plants. Examplesof plants of interest include grain crop plants (such as wheat, oat,barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth,and buckwheat); forage crop plants (such as forage grasses and foragedicots including alfalfa, vetch, clover, and the like); oilseed cropplants (such as cotton, safflower, sunflower, soybean, canola, rapeseed,flax, peanuts, and oil palm); tree nuts (such as walnut, cashew,hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm,olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees;vegetable crop plants such as legumes (for example, beans, peas,lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery,carrot, radish, the brassicas (for example, cabbages, kales, mustards,and other leafy brassicas, broccoli, cauliflower, Brussels sprouts,turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons,summer squashes, winter squashes), edible alliums (for example, onions,garlic, leeks, shallots, chives), edible members of the Solanaceae (forexample, tomatoes, eggplants, potatoes, peppers, groundcherries), andedible members of the Chenopodiaceae (for example, beet, chard, spinach,quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits(for example, orange, lime, lemon, grapefruit, and others), stone fruits(for example, apricot, peach, plum, nectarine), banana, pineapple,grape, kiwifruit, papaya, avocado, and berries; plants grown for biomassor biofuel (for example, Miscanthus grasses, switchgrass, jatropha, oilpalm, eukaryotic microalgae such as Botryococcus braunii, Chlorellaspp., and Dunaliella spp., and eukaryotic macroalgae such as Gracilariaspp., and Sargassum spp.); and ornamental plants including ornamentalflowering plants, ornamental trees and shrubs, ornamental groundcovers,and ornamental grasses.

This invention also provides commodity products produced from atransgenic plant cell, plant, or seed of this invention, including, butnot limited to, harvested leaves, roots, shoots, tubers, stems, fruits,seeds, or other parts of a plant, meals, oils, extracts, fermentation ordigestion products, crushed or whole grains or seeds of a plant, or anyfood or non-food product including such commodity products produced froma transgenic plant cell, plant, or seed of this invention. The detectionof one or more of nucleic acid sequences of the recombinant DNAconstructs of this invention in one or more commodity or commodityproducts contemplated herein is de facto evidence that the commodity orcommodity product contains or is derived from a transgenic plant cell,plant, or seed of this invention.

Generally a transgenic plant having in its genome a recombinant DNAconstruct of this invention exhibits increased resistance to aLeptinotarsa species infestation. In various embodiments, for example,where the transgenic plant expresses a recombinant DNA construct of thisinvention that is stacked with other recombinant DNA for impartingadditional traits, the transgenic plant has at least one additionalaltered trait, relative to a plant lacking the recombinant DNAconstruct, selected from the group of traits consisting of:

-   -   (a) improved abiotic stress tolerance;    -   (b) improved biotic stress tolerance;    -   (c) modified primary metabolite composition;    -   (d) modified secondary metabolite composition;    -   (e) modified trace element, carotenoid, or vitamin composition;    -   (f) improved yield;    -   (g) improved ability to use nitrogen, phosphate, or other        nutrients;    -   (h) modified agronomic characteristics;    -   (i) modified growth or reproductive characteristics; and    -   (j) improved harvest, storage, or processing quality.

In some embodiments, the transgenic plant is characterized by: improvedtolerance of abiotic stress (e.g., tolerance of water deficit ordrought, heat, cold, non-optimal nutrient or salt levels, non-optimallight levels) or of biotic stress (e.g., crowding, allelopathy, orwounding); by a modified primary metabolite (e.g., fatty acid, oil,amino acid, protein, sugar, or carbohydrate) composition; a modifiedsecondary metabolite (e.g., alkaloids, terpenoids, polyketides,non-ribosomal peptides, and secondary metabolites of mixed biosyntheticorigin) composition; a modified trace element (e.g., iron, zinc),carotenoid (e.g., beta-carotene, lycopene, lutein, zeaxanthin, or othercarotenoids and xanthophylls), or vitamin (e.g., tocopherols)composition; improved yield (e.g., improved yield under non-stressconditions or improved yield under biotic or abiotic stress); improvedability to use nitrogen, phosphate, or other nutrients; modifiedagronomic characteristics (e.g., delayed ripening; delayed senescence;earlier or later maturity; improved shade tolerance; improved resistanceto root or stalk lodging; improved resistance to “green snap” of stems;modified photoperiod response); modified growth or reproductivecharacteristics (e.g., intentional dwarfing; intentional male sterility,useful, e.g., in improved hybridization procedures; improved vegetativegrowth rate; improved germination; improved male or female fertility);improved harvest, storage, or processing quality (e.g., improvedresistance to pests during storage, improved resistance to breakage,improved appeal to consumers); or any combination of these traits.

In another embodiment, transgenic seed, or seed produced by thetransgenic plant, has modified primary metabolite (e.g., fatty acid,oil, amino acid, protein, sugar, or carbohydrate) composition, amodified secondary metabolite composition, a modified trace element,carotenoid, or vitamin composition, an improved harvest, storage, orprocessing quality, or a combination of these. In another embodiment, itcan be desirable to change levels of native components of the transgenicplant or seed of a transgenic plant, for example, to decrease levels ofan allergenic protein or glycoprotein or of a toxic metabolite.

Generally, screening a population of transgenic plants each regeneratedfrom a transgenic plant cell is performed to identify transgenic plantcells that develop into transgenic plants having the desired trait. Thetransgenic plants are assayed to detect an enhanced trait, e g.,enhanced water use efficiency, enhanced cold tolerance, increased yield,enhanced nitrogen use efficiency, enhanced seed protein, and enhancedseed oil. Screening methods include direct screening for the trait in agreenhouse or field trial or screening for a surrogate trait. Suchanalyses are directed to detecting changes in the chemical composition,biomass, physiological properties, or morphology of the plant. Changesin chemical compositions such as nutritional composition of grain aredetected by analysis of the seed composition and content of protein,free amino acids, oil, free fatty acids, starch, tocopherols, or othernutrients. Changes in growth or biomass characteristics are detected bymeasuring plant height, stem diameter, internode length, root and shootdry weights, and (for grain-producing plants such as maize, rice, orwheat) ear or seed head length and diameter. Changes in physiologicalproperties are identified by evaluating responses to stress conditions,e.g., assays under imposed stress conditions such as water deficit,nitrogen or phosphate deficiency, cold or hot growing conditions,pathogen or insect attack, light deficiency, or increased plant density.Other selection properties include days to flowering, days to pollenshed, days to fruit maturation, fruit or tuber quality or amountproduced, days to silking in maize, leaf extension rate, chlorophyllcontent, leaf temperature, stand, seedling vigor, internode length,plant height, leaf number, leaf area, tittering, brace roots, stayinggreen, stalk lodging, root lodging, plant health, fertility, green snap,and pest resistance. In addition, phenotypic characteristics ofharvested fruit, seeds, or tubers can be evaluated; for example, intomato and eggplant this can include the total number or weight of fruitharvested or the color, acidity, sugar content, or flavor of such fruit,and in potato this can include the number or total weight of tubersharvested and the quality of such tubers.

Specific assays with the compositions and methods of this invention canbe carried out in solanaceous plants including potato, tomato, eggplant,and peppers, either as hybrids or inbreds; such assays are useful, e.g.,for identifying or selecting plants with improved resistance to Coloradopotato beetle (larvae or adults), for determining insecticidallyeffective amounts of a given composition, or for determining effectivetreatment regimes. Non-limiting examples of such assays include thefollowing.

An in planta Colorado potato beetle (larvae or adults) assay is carriedout in tomato plants with 6 replicates per treatment. Big Cherry tomatoplants are seeded in Readi-Earth soil containing 6 pounds/cubic yard14-14-14 fertilizer and maintained in a 27 degree Celsius, 50% relativehumidity growth chamber for three weeks. On the day of the assay,double-stranded RNA is diluted into 25 milliliters of spray solution (20millimolar sodium phosphate buffer (pH 6.8), optionally containing asurfactant, e.g., 0.2% Silwet L77) to the desired concentration, andapplied to the plants using a track sprayer at a rate of 15 gallons peracre. A higher concentration (e.g., 100 micrograms/milliliter) can beused for initially assaying a polynucleotide for activity, and lowerconcentrations (e.g., between about 0.1 to about 1 microgram permilliliter) can be used in subsequent assays such as those fordetermining relative efficacy of various polynucleotides. Plants arecaged individually with mesh sleeves, and infested with 12 neonatalLeptinotarsa decemlineata (Colorado potato beetle) larvae. Infestedplants are incubated in the growth chamber (27 degrees Celsius, 50%relative humidity) for 12-14 days. At the end of this period, plants areevaluated for level of defoliation, rated as “percent control”, andinsects are collected from plants and soil to evaluate “percent viableinsects recovered” and “average weight of viable insects recovered”.

An in planta Colorado potato beetle (larvae or adults) assay is carriedout in potato plants with 9 replicates per treatment. Cuttings areprepared from mature Atlantic potato plants by cutting the stem at anangle below the second node from the youngest growth. The cutting isdipped into rooting hormone (Rhizopon #1, 0.1% IBA) and immediatelyinserted into pre-wet Readi-Earth soil containing 6 pounds/cubic yard14-14-14 fertilizer. Flats of cuttings are covered to decrease lightexposure and placed in a sealed plastic bag to increase humidity. Overthe next week, the cover is removed and flats are removed from theplastic bags. Plants that are 6-9 inches tall (usually 3 weeks fromcutting date) are used in the assay. On the day of the assay,double-stranded RNA is diluted into 25 milliliters of spray solution (20millimolar sodium phosphate buffer (pH 6.8), optionally containing asurfactant, e.g., 0.2% Silwet L77) to the desired concentration, andapplied to the plants using a track sprayer at a rate of 15 gallons peracre. A higher concentration (e.g., 100 micrograms/milliliter) can beused for initially assaying a polynucleotide for activity, and lowerconcentrations (e.g., between about 0.1 to about 1 microgram permilliliter) can be used in subsequent assays such as those fordetermining relative efficacy of various polynucleotides. Plants arecaged individually with mesh sleeves, and infested with 6 neonatalLeptinotarsa decemlineata (Colorado potato beetle) larvae. Infestedplants are incubated in the growth chamber (27 degree Celsius, 50%relative humidity) for 12-14 days. At the end of this period, plants areevaluated for level of defoliation, rated as “percent control”, andinsects are collected from plants and soil to evaluate “percent viableinsects recovered” and “average weight of viable insects recovered”.

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

EXAMPLES Example 1: Generation of Leptinotarsa cDNA Library

A cDNA library was generated from Leptinotarsa decemlineata (Coloradopotato beetle, “CPB”) neonate larvae, as follows. Total RNA was isolatedfrom 800 third instar Leptinotarsa decemlineata larvae (whole body)using an Ambion Totally RNA isolation kit (catalogue number AM1910, LifeTechnologies, Carlsbad, Calif.) with the optional LiCL precipitationprocedure. PolyA RNA was isolated using Ambion MicroPoly(A) Purist(catalogue number AM1919, Life Technologies, Carlsbad, Calif.). Randomprimed cDNA synthesis was performed using a Superscript Double-StrandedcDNA synthesis kit (catalogue number 11917-010, Life Technologies,Carlsbad, Calif.) with a random hexamer kit (catalogue number 12328-032,Life Technologies, Carlsbad, Calif.). The cDNA library was obtained byhigh-throughput sequencing using commercially available 454 technology(454 Life Sciences, 15 Commercial St., Branford, Conn. 06405, USA), asdescribed in Margulies et al. (2005) Nature, 437:376-380. This provided1,446,014 reads (averaging ˜350 base-pairs in length), which weresupplemented with publicly available Leptinotarsa decemlineata sequencedata from NCBI (including 8,835 expressed sequence tag sequences, 150full-length cDNAs, 839,061 high-throughput DNA and RNA archived sequencereads) to provide a total of 2294087 combined reads. The combinedsequence data were assembled into contigs de novo using the Newbler(version 2.3) software package (454 Life Sciences, 15 Commercial St.,Branford, Conn. 06405, USA). Approximately 38,164 assembled contigs wereidentified from the sequence data.

Example 2: Selection of Low-Copy Target Genes

Leptinotarsa target gene sequences predicted to be effective targets forRNAi-mediated silencing were identified as follows. Low-copy genes, andin particular single-copy genes, were selected as targets forRNAi-mediated silencing as these genea are unlikely to have theirfunction recapitulated by a paralogue. A public database of orthologousgenes, OrthoDB6 (available at cegg.unige.ch/orthodb6 and described inWaterhouse et al. (2012) Nucleic Acids Res., PMID:23180791; doi:10.1093/nar/gks1116) was filtered to select a subset of 766 genes thatwere single-copy or low-copy in Tribolium castaneum (red flour beetle, acoleopteran species) as well as single-copy or low-copy in all availablearthropod genomes in the database (at the time this application is filed33 other arthropod genomes were available). Tribolium castaneum is acoleopteran species and is therefore closely related to Leptinotarsa,which makes it likely that a single-copy or low-copy gene present in theTribolium castaneum genome database will also be a single-copy orlow-copy gene in the Leptinotarsa decemlineata genome, at least forgenes that have high sequence similarity between the two organisms. Fromthe 38,164 unigenes obtained from the Leptinotarsa decemlineata(Colorado potato beetle, CPB) sequencing and assembly described inExample 1, a subset of 725 genes were identified using a translatednucleotide BLAST search (tblastx) as genes having high sequencesimilarity (significance or e-value of less than or equal to 1×10⁻¹⁵) tothe 766 single-copy or low-copy Tribolium castaneum genes in the OrthoDBdatabase.

For sequence annotation, SmartBlast annotation was performed by usingNCBI's Blastall 2.2.21 software to search Leptinotarsa decemlineatacontigs against the publicly available uniref90.fasta database(ftp.uniprot.org/pub/databases/uniprot/current_release/unirefuniref90/).The blast search was performed in blastx mode (translated Leptinotarsadecemlineata nucleotide queries searched against the uniref90 proteindatabase). Only blast hits with an e-value less than or equal to 9e-9were retained. For each Leptinotarsa decemlineata contig the descriptionline from the uniref90 best hit was used as an annotation. When noSmartBlast hits were found, the sequence was subjected to asupplementary Pfam search. To accomplish this, the longest open readingframe (ORF) was identified for each Leptinotarsa decemlineata contig andused to query the publicly available Pfam-A database(ftp.sanger.ac.uk/pub/databases/Pfam/current_release) using the publiclyavailable HMMER 3.0 software package (hmmer.janelia.org/). Leptinotarsadecemlineata contigs with a Pfam hit with an e-value less than or equalto 1e-5 were annotated with the protein family name and the Pfamidentifier. Leptinotarsa decemlineata contigs with no SmartBlast or Pfamhit were annotated as “novel protein”.

The 725 Leptinotarsa decemlineata genes identified as having highsequence similarity to single-copy or low-copy Tribolium castaneum genesas described above are provided as SEQ ID NOs:1-725, with each geneannotated based on sequence similarity to Tribolium castaneum and/orOrthoDB sequences, or by conserved Pfam domains. For each Leptinotarsadecemlineata gene, the homologous Tribolium castaneum gene is alsoidentified in the annotation, together with the similarity e-value foreach pair.

Example 3: Selection of Leptinotarsa Target Genes

cDNA sequences corresponding to useful target genes for controllingLeptinotarsa species by RNAi-mediated silencing were selected from thesequences obtained from the Leptinotarsa decemlineata (Colorado potatobeetle, CPB) sequencing and assembly described in Example 1. This subsetof cDNA sequences or target genes is provided in SEQ ID NOs:726-830. Itis recognized that analogous sequences can be obtained from any otherLeptinotarsa species referred to herein.

Example 4: Selection of Polynucleotide Triggers by “Tiling”

One non-limiting example of a method for selecting a polynucleotidetrigger for expression in a transgenic plant or use in a composition fortopical application to the surface of a transgenic or non-transgenicplant involves the mapping of efficacious polynucleotide sequences (orsegments of sequences) using a whole-gene (or full-length referencesequence) tiling array approach. Sequences selected from SEQ IDNOs:1-725 and SEQ ID NOs:726-830 and SEQ ID NOs:1087-1094 are dividedinto “tiling sequences” or segments of 200-300 contiguous nucleotidesalong the entire length of the selected target sequence. The tilingsequences can be designed to be contiguous segments of the selectedsequence with no overlap or to overlap about 18, 19, 20, 21, 22, 23, 24or 25 nucleotides in adjacent segments of the selected sequence.Polynucleotide triggers corresponding to each 200-300 nucleotide tilingsequence (in sense, anti-sense, or both sense and anti-senseorientation) are synthesized for efficacy screening.

The polynucleotide triggers are tested by any convenient means forefficacy in silencing the Leptinotarsa species target gene. An exampleof a suitable test is a diet bioassay such as that described in Examples5 and 6. Another suitable test involves the topical application of thepolynucleotide triggers either directly to Leptinotarsa individuals orto the surface of a plant to be protected from a Leptinotarsa speciesinfestation. One desired result of treatment with a polynucleotidetrigger is prevention or control of a Leptinotarsa species infestation,e.g., by inducing in a Leptinotarsa insect a physiological orbehavioural change such as, but not limited to, growth stunting,increased mortality, decrease in reproductive capacity, decrease in orcessation of feeding behavior or movement, or decrease in or cessationof metamorphosis stage development. Another desired result of treatmentwith a polynucleotide trigger is provision of a solanaceous plant thatexhibits improved resistance to a Leptinotarsa species infestation, suchas a potato, tomato, eggplant, or pepper plant that exhibits improvedresistance to an infestation by Leptinotarsa decemlineata (Coloradopotato beetle, CPB) or other Leptinotarsa species. Polynucleotidetriggers may be screened in sets. For example, sets of five individualpolynucleotide triggers are pooled into a single polynucleotidecomposition and topically applied to plants. Those sets showing betterefficacy are then re-screened by testing the individual componentpolynucleotide triggers for efficacy.

The tiling procedure can be repeated, if desired. A polynucleotidetrigger found to provide desired activity can itself be tiled. Theparent polynucleotide trigger is divided into smaller overlapping ornon-overlapping segments along the length of the parent polynucleotidetrigger. For example, the parent polynucleotide trigger is divided intosegments of 50-60 nucleotides in length along the entire length of theparent polynucleotide trigger. Polynucleotide triggers corresponding toeach 50-60 nucleotide tiling sequence (in sense, anti-sense, or bothsense and anti-sense orientation) are synthesized for efficacyscreening. Additional rounds of tiling analysis can be carried out,where triggers as short as 18,19, 20, 21, 22, 23, 24, or 25 nucleotidesare tested.

Effective polynucleotide triggers of any size are used to make acomposition for topical application or a recombinant DNA constructuseful for making a transgenic plant.

Example 5

This example illustrates a non-limiting assay useful for evaluating theLeptinotarsa-controlling efficacy of polynucleotide triggers. Morespecifically, this example illustrates double-stranded RNA triggerscomprising a nucleotide sequence that is complementary to at least 21contiguous nucleotides of a Leptinotarsa target gene (e.g., a targetgene selected from the Target Gene Sequences Group, or having a DNAsequence selected from the group consisting of: SEQ ID NOs:1-725 and SEQID NOs:726-830 and SEQ ID NOs:1087-1094, or the DNA complement thereof),and a bioassay useful for evaluating the Leptinotarsa-controllingefficacy of these dsRNA triggers.

Triggers of between about 50 to about 500 base-pairs (more specifically,of between about 100 to about 450 base-pairs) in length were designedfor Leptinotarsa target genes (see Examples 2 and 3). Blunt-endeddouble-stranded RNAs (dsRNAs) with the anti-sense strand sequencesprovided in SEQ ID NOs: 831-1085 were manufactured for the target geneslisted in Table 1.

The dsRNA triggers (Table 1) for suppressing the Leptinotarsa targetgenes were tested using the following methodology to assay mortality orstunting of Leptinotarsa decemlineata larvae due to contact with oringestion of the polynucleotide triggers. Bioassays with the Coloradopotato beetle (CPB), Leptinotarsa decemlineata, were conducted using anartificial diet consisting of 13.2 grams/liter agar (Serva 11393), 140.3grams/liter Bio-Serve pre-mix (F9380B), 5 milliliters/liter KOH (18.3%w/w), and 1.25 milliliters/liter formalin (37%). The diet was dispensedin 200 microliter aliquots into 96-well plates and dried briefly priorto sample application. Twenty microliters of test sample were appliedper well, with sterile water serving as the untreated control (UTC).Plates were allowed to dry before adding insect larvae. One neonate CPBlarva was added per well with a fine paintbrush. Plates were sealed withMylar and ventilated using an insect pin. Thirty-two larvae were testedper treatment. The bioassay plates were incubated at 27 degrees Celsius,60% relative humidity, in complete darkness for 10-12 days. The plateswere scored for larval stunting and mortality. Data was analyzed usingJMP©4 statistical software (SAS Institute, 1995) and a full factorialANOVA was conducted with a Dunnet's test to look for treatment effectscompared to the untreated control (P<0.05). A Tukey-Kramer post hoc testwas performed to compare all pairs of the treatments (P<0.05). Resultsare provided in Table 1.

TABLE 1 SEQ ID dsRNA SEQ NO. OF CPB Diet concen- ID TARGET Bioassaytration Exon NO.* Target Gene GENE Results** (ppm) No. 831 26Sproteasome non-ATPase regulatory subunit 1 825 (+) 0.1 1 832 26Sproteasome non-ATPase regulatory subunit 1 825 (−) 0.1 1 833 26Sproteasome non-ATPase regulatory subunit 1 825 (−) 0.1 1 834 26Sproteasome non-ATPase regulatory subunit 1 825 (−) 0.1 1 835 26Sproteasome non-ATPase regulatory subunit 1 825 (−) 0.1 2 836 Actin 821(−) 0.1 1 837 Actin 821 (−) 0.1 1 838 Actin 821 (−) 0.1 1 839 Actin 821(−) 0.1 1 840 Actin 821 (−) 0.1 1 841 Coatomer subunit beta 822 (−) 0.11 842 Coatomer subunit beta 822 (+) 0.1 1 843 Coatomer subunit beta 822NT 0.1 1 844 Coatomer subunit beta 822 NT 0.1 1 845 Coatomer subunitbeta 822 (−) 0.1 1 846 26S proteasome non-ATPase regulatory subunit 2805 (−) 0.1 1 847 26S proteasome non-ATPase regulatory subunit 2 805 (−)0.1 1 848 26S proteasome non-ATPase regulatory subunit 2 805 (−) 0.1 1849 26S proteasome non-ATPase regulatory subunit 2 805 (+) 0.1 1 850 26Sproteasome non-ATPase regulatory subunit 2 805 (−) 0.1   2? 851 26Sproteasome non-ATPase regulatory subunit 12 806 (−) 0.1 1 852 26Sproteasome non-ATPase regulatory subunit 12 806 (−) 0.1 1 853 26Sproteasome non-ATPase regulatory subunit 12 806 NT 0.1   2? 854 26Sproteasome non-ATPase regulatory subunit 12 806 NT 0.1 1 855 26Sproteasome non-ATPase regulatory subunit 12 806 NT 0.1 1 856 Probable26S proteasome non-ATPase regulatory subunit 3 807 (−) 0.1 1 857Probable 26S proteasome non-ATPase regulatory subunit 3 807 (−) 0.1 1858 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT 0.1 1859 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT 0.1 1860 Probable 26S proteasome non-ATPase regulatory subunit 3 807 NT 0.1 1861 26S proteasome non-ATPase regulatory subunit 7 808 (−) 0.1 1 862 26Sproteasome non-ATPase regulatory subunit 7 808 (−) 0.1 1 863 26Sproteasome non-ATPase regulatory subunit 7 808 NT 0.1 1 884 26Sproteasome non-ATPase regulatory subunit 7 808 NT 0.1 1 865 26Sproteasome non-ATPase regulatory subunit 7 808 NT 0.1 1 866 26Sproteasome non-ATPase regulatory subunit 2 809 (−) 0.1 1 867 26Sproteasome non-ATPase regulatory subunit 2 809 (−) 0.1   2? 868 26Sproteasome non-ATPase regulatory subunit 2 809 (−) 0.1 2 863 26Sproteasome non-ATPase regulatory subunit 2 809 (−) 0.1 1 870 26Sproteasome non-ATPase regulatory subunit 2 809 (−) 0.1 1 871 26Sproteasome non-ATPase regulatory subunit 4 810 (−) 0.1 1 872 26Sproteasome non-ATPase regulatory subunit 4 810 NT 0.1 2 873 26Sproteasome non-ATPase regulatory subunit 4 810 NT 0.1 1 874 26Sproteasome non-ATPase regulatory subunit 4 810 NT 0.1 1 875 26Sproteasome non-ATPase regulatory subunit 4 810 NT 0.1 1 876 26S proteaseregulatory subunit 8 811 NT 0.1 1 877 26S protease regulatory subunit 8811 NT 0.1 1 878 26S protease regulatory subunit 8 811 NT 0.1   2? 87926S protease regulatory subunit 8 811 (−) 0.1 1 880 26S proteaseregulatory subunit 8 811 (−) 0.1 1 881 26S proteasome non-ATPaseregulatory subunit 13 812 NT 0.1 2 882 26S proteasome non-ATPaseregulatory subunit 13 812 NT 0.1 1 883 26S proteasome non-ATPaseregulatory subunit 13 812 (−) 0.1 1 384 26S proteasome non-ATPaseregulatory subunit 13 812 (−) 0.1 1 885 26S proteasome non-ATPaseregulatory subunit 13 812 (−) 0.1 1 886 Putative uncharacterized protein813 NT 0.1 1 887 Putative uncharacterized protein 813 NT 0.1 1 888Putative uncharacterized protein 813 (−) 0.1 1 889 ADP-ribosylationfactor GTPase-activating protein, 814 NT 0.1 1 putative 890ADP-ribosylation factor GTPase-activating protein, 814 NT 0.1 1 putative891 ADP-ribosylation factor GTPase-activating protein, 814 (−) 0.1 1putative 892 ADP-ribosylation factor GTPase-activating protein, 814 (−)0.1 1 putative 893 Golgi-specific brefeldin A-resistance guanine 815 NT0.1 1 nucleotide exchange factor, putative 894 Golgi-specific brefeldinA-resistance guanine 815 NT 0.1 1 nucleotide exchange factor, putative895 Golgi-specific brefeldin A-resistance guanine 815 NT 0.1 1nucleotide exchange factor, putative 896 Golgi-specific brefeldinA-resistance guanine 815 NT 0.1 1 nucleotide exchange factor, putative897 Golgi-specific brefeldin A-resistance guanine 815 NT 0.1   2?nucleotide exchange factor, putative 898 Sec24 protein, putative 816 (+)0.1 2 899 Sec24 protein, putative 816 (−) 0.1   2? 900 Sec24 protein,putative 816 (−) 0.1   2? 901 Sec24 protein, putative 816 (−) 0.1   2?902 Sec24 protein, putative 816 (−) 0.1 1 903 Protein transport proteinSec24B 817 (−) 0.1 1 904 Protein transport protein Sec24B 817 (−) 0.1 1905 Protein transport protein Sec24B 817 (−) 0.1 1 906 Protein transportprotein Sec24B 817 (−) 0.1 1 907 Protein transport protein Sec24B 817(−) 0.1 1 903 Protein transport protein sec31A 813 (−) 0.1 1 909 Proteintransport protein sec31A 818 (−) 0.1 1 910 Protein transport proteinsec31A 818 (+) 0.1 1 911 Protein transport protein sec31A 818 (−) 0.1  2? 912 Protein transport protein sec31A 818 (−) 0.1 1 913 GTP-bindingprotein SAR1B 819 (−) 0.1 1 914 GTP-binding protein SAR1B 819 (−) 0.1 1915 GTP-binding protein SAR1B 819 NT 0.1 2 916 GTP-binding protein SAR1B819 NT 0.1 1 917 GTP-binding protein SAR1B 819 NT 0.1 1 918 Proteintransport protein sec13 820 (−) 0.1 2 919 Protein transport proteinsec13 820 (−) 0.1 1 920 Protein transport protein sec13 820 (−) 0.1 1921 Protein transport protein sec13 820 (−) 0.1 1 922 Ribosomal proteinL13A 741 NT 1.0 2 923 Ribosomal protein L13A 741 NT 1.0 2 924 60Sribosomal protein L5 728 NT 1.0 2 925 60S ribosomal protein L5 728 (+)1.0   2? 926 Ribosomal protein S7 776 NT 1.0 1 927 Ribosomal protein S7776 (−) 1.0 1 928 Ribosomal protein L9 735 (+) 1.0 2 929 Ribosomalprotein L9 735 NT 1.0 1 930 Ribosomal protein L3 726 NT 1.0 2 931Ribosomal protein L3 726 (+) 1.0 2 932 60S ribosomal protein L32 755 (+)1.0 3 933 Ribosomal protein L8 734 NT 1.0 2 934 Ribosomal protein L8 734NT 1.0 2 935 Ribosomal protein S15 785 NT 1.0 2 936 Ribosomal proteinS15 785 NT 1.0 2 937 Ribosomal protein L7A 732 (+) 1.0 3 938 Ribosomalprotein L7A 732 (+) 1.0 3 939 40S ribosomal protein S14 784 NT 1.0 2 94040S ribosomal protein S14 784 (+) 1.0 2 941 40S ribosomal protein S24796 (+) 1.0   2? 942 60S ribosomal protein L10A 737 (+) 1.0 1 943Ribosomal protein L13 740 (+) 1.0 1 944 Ribosomal protein L13 740 (+)1.0 1 945 Ribosomal protein S13 783 (+) 1.0 3 946 Ribosomal protein S13783 NT 1.0 2 947 Ribosomal protein L4e 727 (+) 1.0 3 948 Ribosomalprotein L4e 727 (+) 1.0 2 949 Ribosomal protein S30 803 (+) 1.0 2 950Ribosomal protein S30 803 (+) 1.0 2 951 Ribosomal protein L26 749 (+)1.0   2? 952 Ribosomal protein L26 749 (+) 1.0   2? 953 Ribosomalprotein L31 754 NT 1.0 3 954 60S Ribosomal protein L10 736 NT 1.0 2 95560S Ribosomal protein L10 736 (+) 1.0 2 956 Ribosomal protein S4 772 (+)1.0 3 957 Ribosomal protein S4 772 (+) 1.0 2 958 Ribosomal protein L11e738 (+) 1.0 2 959 Ribosomal protein S6 774 (−) 1.0 1 960 Ribosomalprotein S11 782 (+) 1.0 3 961 Ribosomal protein S11 782 (+) 1.0 3 962Ribosomal protein S11 781 NT 1.0 3 963 Ribosomal protein S11 781 NT 1.03 964 Ribosomal protein L12e 739 (+) 1.0 2 965 Ribosomal protein L12e739 NT 1.0 2 966 Ribosomal protein S5 773 (+) 1.0 2 967 Ribosomalprotein S5 773 (+) 1.0 3 968 Ribosomal protein S18 790 (+) 1.0 2 969Ribosomal protein S18 790 (+) 1.0 2 970 Ribosomal protein L23A 747 (+)1.0 2 971 Ribosomal protein L23A 747 (+) 1.0 2 972 Ribosomal proteinL35A 759 NT 1.0 1 973 Ribosomal protein L35A 759 (+) 1.0 2 974 Ribosomalprotein L21 746 NT 1.0   2? 975 Ribosomal protein L21 746 NT 1.0   2?976 Ribosomal protein L21 745 (+) 1.0 1 977 Ribosomal protein L21 745(−) 1.0   2? 978 Ribosomal protein S8 777 (+) 1.0 2 979 Ribosomalprotein S8 777 (+) 1.0 3 980 Ribosomal protein S16 788 NT 1.0 1 981Ribosomal protein S16 799 NT 1.0 2 982 Ribosomal protein L18Ae 744 (+)1.0 2 983 Ribosomal protein S6 775 (+) 1.0 1 984 Ribosomal protein S3768 NT 1.0 2 985 Ribosomal protein S3 768 (+) 1.0 2 986 Ribosomalprotein S17 789 NT 1.0 2 987 Ribosomal protein S15A 786 (+) 1.0 2 988Ribosomal protein L7 730 (+) 1.0   2? 989 Ribosomal protein L7 730 (+)1.0 2 990 Ribosomal protein S4 771 NT 1.0 2 991 Ribosomal protein S4 771(+) 1.0 2 992 40S ribosomal protein S3A 769 (+) 1.0 1 993 40S ribosomalprotein S3A 769 NT 1.0 1 994 Ribosomal protein L36 760 (+) 1.0 1 995Ribosomal protein L37 762 (+) 1.0 2 996 Ribosomal protein L37 763 (+)1.0 2 997 Ribosomal protein S19 792 (+) 1.0 1 998 Ribosomal protein S19792 NT 1.0 1 999 Ribosomal protein S19 792 (+) 1.0 1 1000 Ribosomalprotein S20 794 NT 1.0 1 1001 Ribosomal protein L15 743 NT 1.0 2 1002Ribosomal protein L35A 758 NT 1.0 1 1003 Ribosomal protein L35A 758 NT1.0 1 1004 40S ribosomal protein S21 795 NT 1.0 3 1005 Ribosomal proteinS29 802 NT 1.0 1 1006 Ribosomal protein S8 778 (+) 1.0 1 1007 40Sribosomal protein S3A 770 (+) 1.0 1 1008 Ribosomal protein L24 748 (+)1.0 2 1009 Ribosomal protein S16 787 (+) 1.0 2 1010 Ribosomal proteinL7A 733 (+) 1.0 1 1011 40S ribosomal protein S9 780 NT 1.0 2 1012 40Sribosomal protein SA 804 NT 1.0 1 1013 40S ribosomal protein SA 804 (+)1.0 1 1014 Ribosomal protein L37Ae 764 (−) 1.0   2? 1015 60S Ribosomalprotein L23 797 NT 1.0 1 1016 Ribosomal protein L7 731 NT 1.0 2 1017Ribosomal protein L36 761 NT 1.0 1 1018 40S ribosomal protein S9 779 (+)1.0   2? 1019 Ribosomal protein S26 798 (+) 1.0 3 1020 Ribosomal proteinL34A 756 (+) 1.0 2 1021 Ribosomal protein L27Ae 751 NT 1.0 1 1022Ribosomal protein L27Ae 751 (+) 1.0 1 1023 40S ribosomal protein S28 801(−) 1.0   2? 1024 Ribosomal protein L29 753 (−) 1.0 3 1025 Ribosomalprotein L28 752 (+) 1.0 4 1026 Ribosomal protein L28 752 NT 1.0 4 1027Ribosomal biogenesis protein RLP24 765 NT 1.0 2 1028 Ribosomalbiogenesis protein RLP24 765 (−) 1.0 1 1029 Ribosomal protein L27 750(+) 1.0 2 1030 Ribosomal protein L27 750 (+) 1.0 2 1031 39S ribosomalprotein L13 766 (−) 1.0 3 1032 39S ribosomal protein L13 766 (−) 1.0 31033 Ribosomal protein S2 767 (+) 1.0 1 1034 40S ribosomal protein S28800 (−) 1.0   2? 1035 Ribosomal protein L14 742 (+) 1.0 2 1036 Ribosomalprotein L6 729 (+) 1.0 2 1037 Coatomer subunit beta 822 (+) 1.0 2 1038Coatomer subunit gamma 828 (+) 1.0 2 1039 Myosin VIIa 824 (+) 1.0 2 1040Myosin VIIa 823 (+) 1.0 1 1041 Actin 821 (+) 1.0 1 1042 26S proteasomenon-ATPase regulatory subunit 1 826 (+) 1.0 2 1043 26S proteasomenon-ATPase regulatory subunit 1 825 (+) 1.0 2 1044 crooked neck 830 NT1.0 1 1045 crooked neck 829 (+) 1.0 2 1046 Predicted putative protein827 (+) 1.0 2 1047 26S proteasome non-ATPase regulatory subunit 2 805(+) 1.0 2 1048 26S proteasome non-ATPase regulatory subunit, putative806 (−) 1.0 2 1049 Probable 26S proteasome non-ATPase regulatory subunit3 807 (+) 1.0 1 1050 26S proteasome non-ATPase regulatory subunit 7 803(+) 1.0 2 1051 26S proteasome non-ATPase regulatory subunit 2 809 NT 1.02 1052 26S proteasome non-ATPase regulatory subunit 4 810 (−) 1.0 3 105326S protease regulatory subunit 8 811 (+) 1.0 3 1054 26S proteasomenon-ATPase regulatory subunit 13 812 (+) 1.0 3 1055 Putativeuncharacterized protein 813 (−) 1.0 2 1056 ADP-ribosylation factorGTPase-activating protein, 814 (−) 1.0 2 putative 1057 Golgi-specificbrefeldin A-resistance guanine 815 (−) 1.0   2? nucleotide exchangefactor, putative 1058 Sec24 protein, putative 816 (+) 1.0 2 1059 Proteintransport protein Sec24B 817 (−) 1.0 1 1060 Protein transport proteinsec31A 818 (+) 1.0 2 1061 GTP-binding protein SAR1B 819 (+) 1.0 2 1062Protein transport protein sec13 820 (−) 1.0   2? 1063 Sec24B protein 817(−) 1.0 1 1064 Coatomer submit beta 822 (+) 1.0 2 1065 Coatomer subunitgamma 828 (+) 1.0 2 1066 Myosin VIIa 824 (+) 1.0 2 1067 Myosin VIIa 823(+) 1.0 2 1068 Actin 821 (+) 1.0 1 1069 26S proteasome non-ATPaseregulatory subunit 1 825 NT 1.0 2 1070 Crooked neck 829 (+) 1.0 2 107126S proteasome non-ATPase regulatory subunit 2 805 (−) 1.0 2 1072 26Sproteasome non-ATPase regulatory subunit 12 806 (−) 1.0 2 1073 Probable26S proteasome non-ATPase regulatory subunit 3 807 (+) 1.0 1 1074 26Sproteasome non-ATPase regulatory subunit 7 808 (+) 1.0 2 1075 26Sproteasome non-ATPase regulatory subunit 2 809 (+) 1.0 2 1076 26Sproteasome non-ATPase regulatory subunit 4 810 (−) 1.0 2 1077 26Sprotease regulatory subunit 8 811 (+) 1.0 3 1078 26S proteasomenon-ATPase regulatory subunit 13 812 (+) 1.0 3 1079 ADP-ribosylationfactor GTPase-activating protein, 814 (−) 1.0 2 putative 1080Golgi-specific brefeldin A-resistance guanine 815 (+) 1.0 1 nucleotideexchange factor, putative 1081 Sec24 protein, putative 816 (+) 1.0 11082 Protein transport protein Sec24B 817 (+) 1.0 1 1083 Proteintransport protein sec31A 818 (−) 1.0 1 1084 GTP-binding protein SAR1B819 (+) 1.0 1 1085 Protein transport protein sec13 820 (+) 1.0 1*sequence of anti-sense strand of the dsRNA trigger **(+) significantstunting or mortality compared with water-treated control; (−) nosignificant stunting or mortality compared with water-treated control;NT = either (1) trigger was not tested, or (2) both of the followingoccurred: the sample did not provide significant stunting/mortality andthe positive control did not provide significant stunting mortality inthat test. Positive control used in this assay was the dsRNA triggertargeting beta coatomer and having the sense strand sequence of SEQ IDNO: 1086, previously disclosed as SEQ ID NO: 880 in U.S. Pat. No.7,943,819.

Where available genomic sequence data permitted, the number of exonsspanned by a given trigger sequence was determined and is provided inTable 1: “1” indicates the trigger sequence appears to be contained in asingle contiguous genomic locus; “2?” indicates that the full length ofthe trigger did not align to the genome, with at least 40 base-pairsmissing, which may indicate incompleteness of the available genomicsequence data.

Additional cDNA sequences encoding subunits of a Leptinotarsadecemlineata (Colorado potato beetle, CPB) exocyst complex wereidentified from a separate sequencing and assembly project asLeptinotarsa target genes. These Leptinotarsa exocyst target genes, SEQID NOs:1087-1094, are useful in designing polynucleotide triggerscomprising at least 21 contiguous nucleotides complementary to anexocyst target gene and useful for controlling Leptinotarsa speciesinfestations, and in making transgenic plants expressing suchpolynucleotide triggers for resistance to Leptinotarsa speciesinfestations.

Triggers of between about 50 to about 500 base-pairs (more specifically,of between about 100 to about 450 base-pairs) in length are designed foreach of the Leptinotarsa exocyst target genes (SEQ ID NOs:1087-1094) asdescribed in Example 4. These triggers are tested using the samemethodology as that described above for the polynucleotides in Table 1.

In a non-limiting example, a polynucleotide trigger, designed to targetthe Leptinotarsa decemlineata Exo70 gene (SEQ ID NO:1093), was producedas a blunt-ended double-stranded RNA having the anti-sense strandsequence of SEQ ID NO:1095. This trigger gave significant stunting andsignificant mortality at both concentrations tested, using themethodology described above. Results are provided in Table 2.

TABLE 2 SEQ ID SEQ Trigger NO. OF CPB Diet dsRNA ID Length TARGETBioassay concentration NO.* (bp) Target Gene GENE Results** (ppm) 1095277 Exo70 1093 (+) 0.1 1095 277 Exo70 1093 (+) 0.033 *sequence ofanti-sense strand of the dsRNA trigger **(+) significant stunting ormortality compared with water-treated control; (−) no significantstunting or mortality compared with water-treated control; NT = either(1) trigger was not tested, or (2) both of the following occurred: thesample did not provide significant stunting/mortality and the positivecontrol did not provide significant stunting/mortality in that test.Positive control used in this assay was the dsRNA trigger targettingbeta coatomer and having the sense strand sequence of SEQ ID NO: 1086,previously disclosed as SEQ ID NO: 880 in U.S. Pat. No. 7,943,819.

Example 6

This example illustrates non-limiting embodiments of polynucleotides ofthis invention, insecticidal compositions for controlling a Leptinotarsaspecies, and a representative assay useful for evaluating theLeptinotarsa-controlling efficacy of such polynucleotides.

Five dsRNA triggers (having anti-sense strand sequences of SEQ IDNOs:989, 1049, 1050, 1078, and 1084; see Table 1) for suppressingLeptinotarsa target genes were tested using the following leaf discmethodologies to assay mortality or stunting of Leptinotarsadecemlineata larvae due to contact with or ingestion of thepolynucleotide triggers.

For the leaf disc bioassay with adult insects, newly emerged Coloradopotato beetle (CPB, Leptinotarsa decemlineata) adults were collected andmaintained on potato foliage for up to 7 days, and then fasted for 6-8hours prior to beginning the bioassay. Fifteen adults per treatment(trigger/dose) were used. Ten microliters containing 250, 83.3, 27.8, or9.3 nanograms of dsRNA trigger in a 0.1% Silwet L77 solution inUltraPure water (Invitrogen) was applied to 15-millimeter-diameterpotato (Atlantic variety) leaf discs; control leaf discs were treatedwith either the formulation 0.1% Silwet L77 solution or with a negativecontrol trigger designed to silence green fluorescent protein (GFP).Treated leaf discs were placed individually into wells of 6-well clusterplates containing 2 milliliters/well of a solidified 2% agaragar/distilled water matrix. A single CPB adult was placed in each welland incubated overnight to allow it to consume the leaf disc; in caseswhere the leaf disc was not totally consumed, the insect was likely deador damaged from handling and was excluded from the assay. The next day,the CPB adults from a given trigger/dose treatment were collectivelytransferred to a feeding arena made from a covered, aerated 16-ouncetranslucent plastic container lined at its base with filter paper andcontaining potato (Atlantic variety) foliage with stems inserted in awater-filled tube for freshness. The insects were incubated in thefeeding arena in an environmental chamber (27 degrees Celsius; 60%relative humidity; 16 hours light/8 hours dark) with potato foliagereplenished as needed. Insect viability was monitored daily. Insectswere recorded as active (viable), moribund (does not return to feetafter 10 seconds after being placed on its back), or dead. Viabilityresults are provided in Table 3.

TABLE 3 CPB Target gene SEQ Days since treatment Treatment ID NO. 5 6 78 9 10 12 14 16 Formulation-1 n/a 100 100 100 100 100 100 100 100 100Formulation-2 n/a 93 93 93 93 93 93 86 86 86 SEQ ID NO. 1115, n/a 100100 100 100 100 100 80 80 60 GFP-1 SEQ ID NO. 1115, n/a 93 93 87 87 8080 80 80 80 GFP-2 SEQ ID NO. 989*, 730 87 87 80 33 0 0 0 0 0 250 ng  SEQID NO. 989*, 730 100 100 79 43 29 7 0 0 0 83 ng SEQ ID NO. 989*, 730 100100 80 47 27 0 0 0 0 28 ng SEQ ID NO. 989*, 730 93 93 73 60 33 0 0 0 0 9 ng SEQ ID NO. 1049*, 807 40 13 0 0 0 0 0 0 0 250 ng  SEQ ID NO.1049*, 807 80 7 7 7 0 0 0 0 0 83 ng SEQ ID NO. 1049*, 807 80 13 13 13 137 13 7 7 28 ng SEQ ID NO. 1049*, 807 87 73 60 60 60 60 53 53 53  9 ngSEQ ID NO. 1050*, 808 60 13 0 0 0 0 0 0 0 250 ng  SEQ ID NO. 1050*, 80860 20 0 0 0 0 0 0 0 83 ng SEQ ID NO. 1050*, 808 86 29 29 14 14 14 14 1414 28 ng SEQ ID NO. 1050*, 808 80 60 60 53 53 53 47 40 40  9 ng SEQ IDNO. 1078*, 812 67 27 20 0 0 0 0 0 0 250 ng  SEQ ID NO. 1078*, 812 60 137 7 7 7 7 7 7 83 ng SEQ ID NO. 1078*, 812 73 33 20 13 13 13 13 13 13 28ng SEQ ID NO. 1078*, 812 100 80 80 67 60 60 53 47 47  9 ng SEQ ID NO.1084*, 819 33 0 0 0 0 0 0 0 0 250 ng  SEQ ID NO. 1084*, 819 73 33 7 0 00 0 0 0 83 ng SEQ ID NO. 1084*, 819 73 40 33 33 33 33 20 20 20 28 ng SEQID NO. 1084*, 819 80 60 53 53 53 53 47 47 40  9 ng *sequence ofanti-sense strand of the dsRNA trigger, unless otherwise noted.“Formulation-1” and “Formulation-2” are duplicates of a null control(0.1% Silwet in water). “GFP-1” and “GFP-2” are duplicates of a negativecontrol using a 377 bp dsRNA trigger targetting green fluorescentprotein (GFP) and having the sense strand sequence of SEQ ID NO: 1115.“n/a” = not applicable.

For the leaf disc bioassay with larvae, neonate Colorado potato beetle(CPB, Leptinotarsa decemlineata) larvae hatched within 24 hours of thebioassay were used. Sixteen larvae per treatment (trigger/dose) wereused. Two microliters containing 250, 83.3, 27.8, or 9.3 nanograms ofdsRNA trigger in a 0.1% Silwet L77 solution in UltraPure water(Invitrogen) was applied to 7-millimeter-diameter potato (Atlanticvariety) leaf discs; control leaf discs were treated with either theformulation 0.1% Silwet L77 solution or with a negative control triggerdesigned to silence green fluorescent protein (GFP). Treated leaf discswere placed individually into wells of 128-well cluster platescontaining 0.5 milliliters/well of a solidified 2% agar agar/distilledwater matrix. A single CPB neonate was placed in each well and incubatedovernight to allow it to consume the leaf disc; in cases where the leafdisc was not totally consumed, the insect was likely dead or damagedfrom handling and was excluded from the assay. The next day, the CPBlarvae from a given trigger/dose treatment were collectively transferredto a feeding arena made from a covered, aerated 16-ounce translucentplastic container lined at its base with filter paper and containingpotato (Atlantic variety) foliage with stems inserted in a water-filledtube for freshness. The insects were incubated in the feeding arena inan environmental chamber (27 degrees Celsius; 60% relative humidity; 16hours light/8 hours dark) with potato foliage replenished as needed.Larval viability was monitored daily. Larvae were recorded as alive ordead. Viability results are provided in Table 4.

TABLE 4 CPB Target gene Days since treatment Treatment SEQ ID NO. 5 6 78 9 10 12 14 16 Formulation-1 n/a 100 100 100 100 100 100 92 54 15Formulation-2 n/a 87 87 87 87 73 73 73 27 20 SEQ ID NO. 1115, GFP- n/a69 69 69 69 69 69 69 50 38 1 SEQ ID NO. 1115, GFP- n/a 100 100 94 94 7575 56 19 19 2 SEQ ID NO. 989*, 250 730 44 38 31 13 13 0 0 0 0 ng SEQ IDNO. 989*, 83 ng 730 19 19 13 0 0 0 0 0 0 SEQ ID NO. 989* 28 ng 730 69 5038 13 13 6 6 6 6 SEQ ID NO. 989*, 9 ng 730 38 13 13 13 6 6 6 6 6 SEQ IDNO. 1049*, 250 807 20 7 7 7 7 0 0 0 0 ng SEQ ID NO. 1049*, 83 807 38 1313 13 13 13 13 13 13 ng SEQ ID NO. 1049*, 28 807 38 13 13 6 6 6 6 6 6 ngSEQ ID NO. 1049*, 9 ng 807 57 21 21 21 21 21 21 21 14 SEQ ID NO. 1050*,250 808 44 31 31 25 19 19 19 0 0 ng SEQ ID NO. 1050*, 83 808 38 19 19 60 0 0 0 0 ng SEQ ID NO. 1050*, 28 808 13 13 13 13 13 13 0 0 0 ng SEQ IDNO. 1050*, 9 ng 808 0 0 0 0 0 0 0 0 0 SEQ ID NO. 1078*, 250 812 19 13 00 0 0 0 0 0 ng SEQ ID NO. 1078*, 83 812 29 14 14 7 7 0 0 0 0 ng SEQ IDNO. 1078*, 28 812 50 31 19 13 13 6 6 0 0 ng SEQ ID NO. 1078*, 9 ng 81260 47 40 27 27 27 27 27 13 SEQ ID NO. 1084*, 250 819 79 43 43 43 29 2121 14 14 ng SEQ ID NO. 1084*, 83 819 56 38 19 19 19 13 13 13 13 ng SEQID NO. 1084*, 28 819 50 38 25 19 19 19 19 19 19 ng SEQ ID NO. 1084*, 9ng 819 75 50 44 44 38 38 38 31 31 *sequence of anti-sense strand of thedsRNA trigger, unless otherwise noted. “Formulation-1” and“Formulation-2” are duplicates of a null control (0.1% Silwet in water).“GFP-1” and “GFP-2” are duplicates of a negative control using a 377 bpdsRNA trigger targetting green fluorescent protein (GFP) and having thesense strand sequence of SEQ ID NO: 1115. “n/a” = not applicable.

Example 7

This example illustrates non-limiting embodiments of polynucleotidetriggers for suppressing Leptinotarsa target genes. More specifically,this example illustrates embodiments of blunt-ended dsRNA triggersconsisting of a sense and a separate anti-sense strand, as well asembodiments of dsRNA triggers in the form of a hairpin (a single RNAtranscript containing both a sense region and an anti-sense region).

Table 5 provides blunt-ended dsRNA triggers with sequences related to a“parent trigger” (see Table 1), where the parent trigger had beendetermined to have insecticidal activity against Leptinotarsadecemlineata (see Tables 1, 3, and 4) and the derivative triggers areblunt-ended dsRNAs corresponding to sub-regions of the parent trigger.

TABLE 5 Target Parent Diet Diet Trigger gene trigger Activity ActivitySEQ ID Target gene SEQ SEQ vs. CPB vs. CPB NO:* name ID NO: ID NO: (0.1ppm) (0.025 ppm) 1096 GTP-binding 819 1084 (−) (−) protein SAR1B 1097GTP-binding 819 1084 (−) (−) protein SAR1B 1098 GTP-binding 819 1084 (+)(−) protein SAR1B 1099 GTP-binding 819 1084 (+) (−) protein SAR1B 1100Probable 26S 807 1049 (−) (−) proteasome non-ATPase regulatory subunit 31101 26S proteasome 808 1050 (−) (−) non-ATPase regulatory subunit 71102 26S proteasome 812 1078 (−) (−) non-ATPase regulatory subunit 131103 Ribosomal 730 989 (−) (−) protein L7 1104 Ribosomal 730 989 (+) (−)protein L7 *sequence of anti-sense strand of the dsRNA trigger

Table 6 provides dsRNA triggers in the form of a hairpin (a single RNAtranscript containing both a sense region and an anti-sense region thathybridize to form dsRNA), with sequences derived from or related to a“parent trigger” (see Table 1), where the parent trigger had beendetermined to have insecticidal activity against Leptinotarsadecemlineata (see Tables 1, 3, and 4). Hairpin triggers are suitable forin vitro expression or in vivo expression when provided in an expressionconstruct with appropriate promoters or other elements to permit theirexpression, e.g., in a bacterial cell or in a plant cell. Thenon-limiting embodiments disclosed in Table 6 each contain a T7 promoter(located at nucleotide positions 1-17 in each hairpin sequence) and a“loop” or spacer located between the sense and the anti-sense regions;the loop contains non-specific (not complementary or identical to anypart of the target gene) nucleotides. One of skill would immediatelyunderstand that the sense and anti-sense regions of the hairpin areuseful in combination with different suitable promoters for expressionin a given cell type, and with different spacer or loop sequences (ornone at all, where nucleotides at the junction of the sense andanti-sense regions form the necessary “turn” or minimal loop in thehairpin). One of skill would also recognize that similar recombinant DNAconstructs are easily designed to encode hairpin dsRNA triggerscorresponding to the blunt-ended dsRNA triggers provided in Tables 1-5or targeting the target genes provided in the Target Gene SequencesGroup.

TABLE 6 nucleotide position of trigger anti- nucleotide nucleotidetrigger anti- sense position of position of Blunt-ended Hairpin senseregion in loop or trigger dsRNA CPB Target Trigger SEQ region inhairpin, spacer in sense region Trigger SEQ Gene SEQ ID NO:* hairpin SEQID NO: hairpin in hairpin ID NO: ID NO: 1105 21-417 1110 418-566567-963   989** 730 1106 21-300 1111 301-450 451-730  1086  1107 21-4531112 454-603 604-1036 1084** 819 1108 21-458 1113 459-608 609-10461050** 808 1109 21-448 1114 449-598 599-1026 1038** 828 *sequence of DNAconstruct encoding the hairpin dsRNA trigger **sequence of anti-sensestrand of the dsRNA trigger SEQ ID NO: 1086 corresponds to the sensestrand sequence of a blunt-ended dsRNA targetting beta coatomer,previously disclosed as SEQ ID NO: 880 in U.S. Pat. No. 7,943,819.

It is anticipated that the combination of certain recombinant RNAs asdescribed herein (e.g., the dsRNA triggers described in Tables 1-6 ortheir hairpin equivalents, or active fragments of these triggers) withone or more non-polynucleotide pesticidal agents will result in asynergetic improvement in prevention or control of Leptinotarsa speciesinfestations, when compared to the effect obtained with the recombinantRNA alone or the non-polynucleotide pesticidal agent alone. Routineinsect bioassays such as the bioassay employing an artificial dietdescribed here are useful for defining dose-responses for larvalmortality or growth inhibition using combinations of the polynucleotidetriggers and one or more non-polynucleotide pesticidal agents (e.g., apatatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensisinsecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdusinsecticidal protein, a Bacillus laterosporous insecticidal protein, anda Bacillus sphaericus insecticidal protein). One of skill in the art cantest combinations of polynucleotides and non-polynucleotide pesticidalagents in routine bioassays to identify combinations of bioactives thatare synergistic and desirable for use in protecting plants fromLeptinotarsa species infestations.

Example 8: Field Efficacy of RNAi-Mediated Control of Leptinotarsadecemlineata

A field trial was performed to test efficacy of topically applied dsRNAtriggers on controlling Leptinotarsa decemlineata (Colorado potatobeetle, CPB) infestations of potato plants under field conditions. ThreedsRNA triggers were tested using topical (foliar spray) application: ablunt-ended dsRNA having an anti-sense strand sequence of SEQ ID NO:989,which targets Ribosomal Protein L7 (encoded by SEQ ID NO: 730); ablunt-ended dsRNA having an anti-sense strand sequence of SEQ IDNO:1049, which targets Probable 26S proteosome non-ATPase regulatorysubunit 3 (encoded by SEQ ID NO: 807); and a hairpin dsRNA encoded bythe DNA construct of SEQ ID NO:1105, which targets Ribosomal Protein L7(encoded by SEQ ID NO: 730). SEQ ID NO:1105 encodes a hairpin dsRNAhaving an anti-sense strand corresponding to SEQ ID NO:989 (see Example7). The experiment was designed with 11 treatments arranged in a randomcomplete block design with four replicates. Test plots consisted ofpotato plants (variety “Superior”) planted in the spring in two 20-footrows with 6-foot row center spacing; plots were maintained according tostandard commercial growing practices. Foliar spray treatments wereperformed twice: a first treatment 36 days after planting and a secondtreatment 43 days after planting. All foliar treatments were appliedwith a 4-nozzle boom equipped with 110003VS spray tips spaced 20 inchesapart, spraying 2 rows at a time, and powered by a carbondioxide-powered backpack sprayer at 40 pounds per square inch,delivering 38 gallons per acre. All life stages of Colorado potatobeetle were recorded for ten randomly selected stems per plot at 3 timepoints: 3 days after the first foliar spray treatment (39 days afterplanting), 7 days after the first foliar spray treatment (43 days afterplanting), and 3 days after the second foliar spray treatment (46 daysafter planting). Defoliation, which is caused primarily by small larvae,was measured at 9 days after the first foliar spray treatment (45 daysafter planting). Two commercial synthetic (small molecule) insecticideswere used as positive controls: Coragen® (chlorantraniliprole, DuPont)and Radiant® (spinetoram, Dow AgroSciences). Results are presented inTable 7; statistically different values are indicated by differentletters (a, b, c, d, e). Those treatments that share a letter, forexample the Untreated Control and 5 grams per acre SEQ ID NO:989Treatment at 3 days after first spray which share the letter “a”, arenot statistically different; while those treatments that do not share aletter, for example the Untreated Control and Coragen® Treatment at 3days after first spray, are statistically different. The effects of thedsRNA triggers increased over time and showed a dose-dependent response;at 3 days after the second foliar spray, all of the dsRNA triggertreatments except for the lowest dose of the dsRNA trigger having ananti-sense strand sequence of SEQ ID NO:1049 resulted in a decrease inlarge larvae that was not significantly different from the syntheticinsecticide positive controls (Coragen® and Radiant Treatments) and thatwas significantly different from the Untreated Control. Defoliation alsoshowed a dose-dependent response to the dsRNA treatments; several of thedsRNA treatments were significantly different from the Untreated Controland all of the dsRNA triggers at the highest dose tested provideddefoliation protection that was not significantly different from thatprovided by the synthetic insecticide positive controls (Coragen® andRadiant Treatments). The decreased number of larvae and decreaseddefoliation or plant damage indicated improved resistance of thedsRNA-treated potato plants to Leptinotarsa decemlineata; these plantswith improved resistance to Leptinotarsa decemlineata are expected toexhibit improved yield (increased harvestable tubers).

TABLE 7 Mean number of Colorado potato beetles/10 stems Large larvaeSmall larvae 3 Rate 3 days 3 days days 3 days (grams after 7 days afterafter 7 days after per first after first second first after first second% Treatment acre) spray spray spray spray spray spray DefoliationUntreated n.a. 115.8 a 201.3 a 72.0 ab 0 45.3 ab 108.0 a  72.5 a ControlSEQ ID 5 63.5 ab 146.5 ab 98.0 ab 3 8.5 bcd  8.3 b 9.8 de NO: 989* SEQID 1 93.3 ab 159.5 a 144.5 a 1.3 33.0 21.8 b 28.8 cd NO: 989* abcd SEQID   0.2 87.8 ab 116.0 118.0 a 0 25.3 33.8 b 45.0 abc NO: 989* abc abcdSEQ ID 5 66.5 ab 135.5 126.0 a 0 2.0 cd 12.8 b 15.0 cde NO: 1049* abcSEQ ID 1 91.0 ab 175.0 a 102.5 0 41.3 abc 33.8 b 32.5 bcd NO: 1049* abUntreated n.a. 115.8 a 201.3 a 72.0 ab 0 45.3 ab 108.0 a  72.5 a ControlSEQ ID   0.2 93.5 ab 113.8 99.3 ab 0.8 59.0 a 80.0 a 68.8 ab NO: 1049*abc SEQ ID 5 61.0 ab 91.3 abc 117.8 a 0 9.0 bcd 14.0 b 12.5 cde NO:1105* SEQ ID 1 72.3 ab 104.8 87.3 ab 0 17.8 bcd  8.8 b 18.8 cd NO: 1105*abc Coragen ®  5** 9.8 b 6.0 c 0.3 b 0 0.0 d  0.0 b 0.0 e Radiant  8**1.3 b 16.8 bc 0.0 b 0 0.5 d  0.0 b 0.0 e P-Value from Anova 0.00530.0004 0.0009 ns 0.0001 <0.0001 <0.0001 n.a., not applicable ns, notsignificant *dsRNA triggers applied in a formulation containing 3milliliters of a commercial spray adjuvant, TACTIC ™ (Loveland Products,Loveland, CO 80538) per 1600 milliliters water **fluid ounces per acre

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of embodiments and illustrative examples, itwill be apparent to those of skill in the art that variations can beapplied to the materials and methods described herein without departingfrom the concept, spirit and scope of this invention. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the spirit, scope and concept of this invention asdefined by the appended claims.

What is claimed is:
 1. A method for controlling a Leptinotarsa speciesinfestation of a plant comprising: (a) contacting said Leptinotarsaspecies with an inhibitory polynucleotide comprising a nucleotidesequence that is complementary to at least 21 contiguous nucleotides ofSEQ ID NO:730, or an RNA transcribed therefrom; or (b) providing in thediet of said Leptinotarsa species an inhibitory polynucleotidecomprising a nucleotide sequence that is complementary to at least 21contiguous nucleotides of SEQ ID NO:730, or an RNA transcribedtherefrom; or (c) causing mortality or stunting in larvae of saidLeptinotarsa species by providing in the diet of said larvae at leastone inhibitory polynucleotide comprising at least one silencing elementcomprising 21 contiguous nucleotides that are complementary to SEQ IDNO:730, or an RNA transcribed therefrom; or (d) topically applying tosaid plant a composition comprising at least one polynucleotidecomprising an inhibitory nucleotide sequence that is complementary to atleast 21 contiguous nucleotides of SEQ ID NO:730, or an RNA transcribedtherefrom; or (e) expressing in said plant at least one inhibitorypolynucleotide comprising at least one segment that is identical orcomplementary to at least 21 contiguous nucleotides of SEQ ID NO:730; or(f) providing to said plant at least one inhibitory polynucleotidecomprising at least one segment that is identical or complementary to atleast 21 contiguous nucleotides of SEQ ID NO:730 or an RNA transcribedtherefrom; or (g) contacting said Leptinotarsa species with aninhibitory polynucleotide comprising at least one segment that isidentical or complementary to at least 21 contiguous nucleotides of SEQID NO:730, or an RNA transcribed therefrom.
 2. The method of claim 1,wherein said inhibitory polynucleotide is a double-stranded RNA.
 3. Themethod of claim 2, wherein said double-stranded RNA is chemicallysynthesized or is produced by expression in a microorganism or byexpression in a plant cell.
 4. The method of claim 2, wherein saiddouble-stranded RNA comprises a strand comprising a sequence selectedfrom the group consisting of: SEQ ID NOs:989, 1104, 1105, and
 1110. 5.The method of claim 1, wherein said method comprises topically applyingto said plant a composition comprising at least one inhibitorypolynucleotide comprising a nucleotide sequence that is complementary toat least 21 contiguous nucleotides of SEQ ID NO:730, or an RNAtranscribed therefrom; and wherein said composition further comprisesone or more components selected from the group consisting of a carrieragent, a surfactant, a cationic lipid, an organosilicone, anorganosilicone surfactant, a polynucleotide herbicidal molecule, anon-polynucleotide herbicidal molecule, a non-polynucleotide pesticide,a safener, and an insect growth regulator.
 6. The method of claim 1,wherein said method comprises contacting said Leptinotarsa species witha solution comprising a double-stranded RNA, wherein at least one strandof the double-stranded RNA is complementary to at least 21 contiguousnucleotides of a gene that encodes a ribosomal protein or an RNAtranscribed from said gene, wherein said Leptinotarsa species isLeptinotarsa decemlineata, and wherein RNA interference is induced andLeptinotarsa decemlineata mortality occurs, and wherein said ribosomalprotein is a ribosomal L7 protein or a protein encoded by SEQ ID NO:730, or wherein said double-stranded RNA comprises a sequence selectedfrom the group consisting of SEQ ID NO: 989, 988, 1104, 1105, or 1110.7. The method of claim 6, wherein said solution further comprises one ormore components selected from the group consisting of an organosiliconesurfactant or a cationic lipid.
 8. The method of claim 1, wherein saidmethod comprises topically applying to said plant a compositioncomprising at least one inhibitory polynucleotide in a manner such thatsaid inhibitory polynucleotide is ingested by Leptinotarsa speciesfeeding on said plant, said inhibitory polynucleotide comprising anucleotide sequence that is complementary to at least 21 contiguousnucleotides of SEQ ID NO:730, or an RNA transcribed therefrom; whereinsaid Leptinotarsa species is Leptinotarsa decemlineata; and wherein saidpolynucleotide is a double-stranded RNA having a strand with a sequenceselected from the group consisting of SEQ ID NO: 989, 988, 1104, 1105 or1110.
 9. The method of claim 1, wherein said Leptinotarsa species isselected from the group consisting of: Leptinotarsa behrensi,Leptinotarsa collinsi, Leptinotarsa decemlineata (Colorado potatobeetle), Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman's greenpotato beetle), Leptinotarsa heydeni, Leptinotarsa juncta (false potatobeetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsapeninsularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsatlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica.
 10. Aplant having improved resistance to a Leptinotarsa species infestation,provided by the method of claim 1, or a fruit, seed, or propagatablepart of said plant, wherein said fruit, seed, or propagatable partcomprises said inhibitory polynucleotide.
 11. The plant of claim 10,wherein said plant is selected from the group consisting of potato,tomato, and eggplant.
 12. A insecticidal composition for controlling aLeptinotarsa species, comprising: (a) at least one double-stranded RNA(dsRNA) comprising at least 21 contiguous nucleotides that arecomplementary to a SEQ ID NO:730, or an RNA transcribed therefrom,wherein said at least one dsRNA causes mortality or stunting of growthin said Leptinotarsa species when ingested or contacted by saidLeptinotarsa species; or (b) at least one dsRNA comprising at least onesilencing element that is complementary to at least 21 contiguousnucleotides of SEQ ID NO:730, wherein said at least one dsRNA causesmortality or stunting of growth in said Leptinotarsa species wheningested or contacted by said Leptinotarsa species; or (c) at least onedsRNA comprising at least one segment that is identical or complementaryto at least 21 contiguous nucleotides of SEQ ID NO:730, or an RNAtranscribed therefrom, wherein said at least one dsRNA causes mortalityor stunting of growth in said Leptinotarsa species when ingested orcontacted by said Leptinotarsa species; or (d) an RNA molecule thatcauses mortality or stunting of growth in a Leptinotarsa species wheningested or contacted by said Leptinotarsa species, wherein said RNAmolecule comprises at least 21 contiguous nucleotides that arecomplementary to SEQ ID NO:730, or an RNA transcribed therefrom; or (e)an insecticidal dsRNA molecule that causes mortality or stunting ofgrowth in a Leptinotarsa species when ingested or contacted by saidLeptinotarsa species, wherein at least one strand of said insecticidaldsRNA molecule comprises 21 contiguous nucleotides that arecomplementary to SEQ ID NO:730, or an RNA transcribed therefrom; or (f)at least one dsRNA comprising a sequence selected from the groupconsisting of: SEQ ID NOs:989, 988, 1104, 1105, and 1110, wherein saidat least one dsRNA causes mortality or stunting of growth in saidLeptinotarsa species when ingested or contacted by said Leptinotarsaspecies.
 13. The insecticidal composition of claim 12, wherein saidinsecticidal composition is in the form of at least one selected fromthe group consisting of a solid, liquid, powder, suspension, emulsion,spray, encapsulation, microbeads, carrier particulates, film, matrix,seed treatment, soil drench, implantable formulation, and in-furrowformulation.
 14. The insecticidal composition of claim 12, furthercomprising at least one component selected from the group consisting ofa carrier agent, a surfactant, a cationic lipid, an organosilicone, anorganosilicone surfactant, a polynucleotide herbicidal molecule, anon-polynucleotide herbicidal molecule, a non-polynucleotide pesticide,a safener, and an insect growth regulator.
 15. The insecticidalcomposition of claim 12, wherein said insecticidal composition comprisesan insecticidal double-stranded RNA molecule that causes mortality orstunting of growth in a Leptinotarsa species when ingested or contactedby said Leptinotarsa species, wherein said insecticidal double-strandedRNA molecule comprises at least one segment that is complementary to 21contiguous nucleotides of a DNA having the sequence of SEQ ID NO:730, oran RNA transcribed from said DNA.
 16. A recombinant DNA constructcomprising a heterologous promoter operably linked to: (a) a DNAencoding an inhibitory nucleotide sequence that is complementary to atleast 21 contiguous nucleotides of SEQ ID NO:730, or an RNA transcribedtherefrom; or (b) a DNA encoding 21 or more contiguous inhibitorynucleotide sequence having 100% identity to a fragment of equivalentlength of a DNA having a sequence of SEQ ID NO:730, or the DNAcomplement thereof; or (c) a DNA encoding at least one silencing elementthat is complementary to at least 21 contiguous nucleotides of SEQ IDNO:730, or an RNA transcribed therefrom; or (d) a DNA encoding at leastone silencing element comprising at least 21 contiguous nucleotides thatare complementary to SEQ ID NO: 730, or an RNA transcribed therefrom; or(e) a DNA encoding a RNA comprising at least 21 contiguous nucleotidesthat are complementary to a nucleotide sequence selected from the groupconsisting of: SEQ ID NOs:989, 988, 1104, 1105, and 1110, or thecomplement thereof; or (f) a DNA encoding a RNA comprising at least onedouble-stranded RNA region, at least one strand of which comprises atleast 21 contiguous nucleotides that are complementary to a nucleotidesequence selected from the group consisting of: SEQ ID NOs:989, 988,1104, 1105, and 1110, or the complement thereat; or (g) a DNA encodingRNA comprising a nucleotide sequence selected from the group consistingof: SEQ ID NOs:989, 988, 1104, 1105, and 1110, or the complementthereof.
 17. A recombinant plant virus vector or a recombinantbaculovirus vector comprising the recombinant DNA construct of claim 16.18. A transgenic solanaceous plant cell having in its genome therecombinant DNA construct of claim
 16. 19. The transgenic solanaceousplant cell of claim 18, wherein said transgenic solanaceous plant cellfurther comprises in its genome a DNA encoding at least one pesticidalagent selected from the group consisting of a patatin, a plant lectin, aphytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBacillus laterosporous insecticidal protein, and a Bacillus sphaericusinsecticidal protein.
 20. A transgenic solanaceous plant comprising thetransgenic solanaceous plant cell of claim 18, or a fruit, seed, orpropagatable part of said transgenic solanaceous plant, wherein saidfruit, seed, or propagatable part comprises said recombinant DNAconstruct.